Endoplasmic reticulum stress as a predictive tool in cancer therapy and a combination therapy for the treatment of cancer

ABSTRACT

Provided are methods, agents and kits for use in assessing the effect of treatment on cancer patients. Further provided is a combination therapy for reducing the administered standard of care doses of anti-cancer agents in treated cancer patients.

SEQUENCE LISTING

The Sequence Listing submitted in text format (.txt) filed on Jul. 31,2018, named “SequenceListing.txt”, created on Jul. 17, 2018 (1.60 KB),is incorporated herein by reference.

TECHNOLOGICAL FIELD

The present disclosure relates to personalized medicine. Morespecifically, the present disclosure provides methods, kits andcompositions for use in predicting the effect of cancer therapy. Inaddition the present disclosure relates to combination therapy forreducing the standard of care doses of an anti-cancer agent in treatedcancer patients.

BACKGROUND ART

References considered to be relevant as background to the presentlydisclosed subject matter are listed below:

-   [1] Mehta, S. et al., 2010, Therapeutic Advances in Medical Oncology    2(2): 125-148.-   [2] Lee, A. S. 2007, Cancer Research 67(8): 3496-3499.-   [3] Zheng, Y. Z. et al., 2014, Breast Cancer Research and Treatment    145: 349-358.-   [4] Sato, M. et al., 2010, Advances in Genetics 69: 97-114.-   [5] Han, K. S. et al., 2015, Oncotarget 6:34818-30.-   [6] WO 2008/042508.-   [7] US 2009/0181472.-   [8] WO 02/082076.-   [9] WO 02/077176.-   [10] WO 2006/046239.-   [11] WO 2007/122622-   [12] WO 2007/091240.-   [13] WO 2008/075349.-   [14] Sandler, U. et al., 2010, Recent advances in clinical medicine,    ISSN: 1790-5125.-   [15] Sandler, U. et al., 2010, J Experimental Therapeutics and    Oncology 8:327-339.-   [16] WO 2015/083167.-   [17] Eisenhauer, E. A. et al., 2009, European Journal of Cancer 45:    228-247.

Acknowledgement of the above references herein is not to be inferred asmeaning that these are in any way relevant to the patentability of thepresently disclosed subject matter.

BACKGROUND

Technological advances greatly increased the understanding of themolecular basis of tumor progression and numerous tumor and treatmentresponse biomarkers have been identified to date (1).

These markers can be generally divided into two types, being prognosticmarkers which aim to objectively evaluate the patient's overall outcome,such as the probability of cancer recurrence after standard treatmentand predictive markers which aim to evaluate the likelihood of benefitfrom a specific clinical intervention (1).

Among the cellular markers implicated with cancer development andprognosis is the glucose-regulated protein GRP78, also referred to asBiP (binding immunoglobulin protein), which primarily resides in theendoplasmic reticulum (ER). GRP78, which belongs to the HSP70 proteinfamily, facilitates proper protein folding, prevents intermediates fromaggregating, and targets misfolded protein for proteasome degradation.In addition, GRP78 serves as an ER stress signaling regulator. GRP78promotes tumor proliferation, survival, metastasis and resistance to awide variety of therapies and thus GRP78 expression may serve as abiomarker for tumor behavior and treatment response (2).

Consistent with the above, cancer cells adapt to chronic stress in thetumor environment by inducing expression of GRP78 (2), which functionsas a potent anti-apoptotic factor and confers drug resistance (3).Indeed, it has been shown that the presence of GRP78 autoantibodies incancer patients' sera is generally associated with poor prognosis (4).GRP78/BiP upregulation following anti-angiogenic therapy has beendemonstrated in multiple studies. For example, Han et al. (5), showedthat sunitinib treatment induced hypoxia in Caki-1 xenografts that wasfollowed by elevated expression of GRP78/BiP in the treated group incomparison to the control group.

Therefore, GRP78 was proposed as a marker for various conditions, interalia for determining whether a subject with cancer is at risk ofdeveloping resistance to hormonal therapy (6), as a prognostic markerfor evaluating tumor grade in the case of head and neck cancer (7) or asa tumor marker of various cancers (8, 9). However, the mechanism bywhich GRP78 acts during cancer progression or cancer treatment is stillnot clear.

A peptide termed “T101” that is encoded by a cDNA unique for the humanthymus was previously identified. This peptide as well as derivativesthereof were implicated, inter alia, for the treatment of cancer via therole of T101 as a stimulator of the immune system (WO 2006/046239, 10).WO 2006/046239 demonstrates that T101 is able to stimulate the immunesystem and to reduce tumor size, suggesting that the peptide affects theproliferation of cancer cells. WO 2006/046239 also suggests animmune-based role for T101, for example in protecting patients duringthe course of standard chemotherapy.

Treatment of cancer by using T101 was also suggested in WO 2007/122622(11), which demonstrates, inter alia, the effect of T101 on thedevelopment of various types of tumors. The peptide T101 was alsodescribed in WO 2007/091240 (12), relating to treatment of immunologicaldiseases and in WO 2008/075349 (13) as well as in the publications bySandler et al. (14 and 15), relating to treating or preventing a diseaseinvolving a cell having T1/ST2 receptor.

In addition, a peptide derivative of T101, termed “Nerofe”, has beenreported to decrease the secretion of proteins that are known to beassociated with cancer metastasis by cancer cells and to directlyinhibit migration of cancer cells in vitro. In addition the peptide wasshown to affect the serum level of vascular endothelial growth factor(VEGF) in cancer patients (WO 2015/083167, 16), and was suggested foruse in a method of preventing or treating cancer metastasis.

GENERAL DESCRIPTION

By one of its aspects the present invention provides a method forpredicting the response of a cancer patient to treatment with anisolated peptide comprising the amino acid sequence denoted by SEQ IDNO. 1 or a functional derivative thereof or a pharmaceuticallyacceptable salt of said isolated peptide, said method comprising thesteps of:

(a) determining the expression level of at least one endoplasmicreticulum (ER) stress marker in at least one biological sample of saidpatient to obtain an expression value, wherein at least one of saidbiological samples is obtained after the initiation of said treatment;(b) determining if the expression value of said at least one ER stressmarker obtained in step (a) is higher or lower with respect to apredetermined standard expression value of said at least one ER stressmarker;wherein an expression value of said at least one ER stress markerobtained in (a) higher than an expression value of said at least one ERstress marker in a predetermined standard indicates that said patient isa responder to said treatment.

In some embodiments, an expression value of said at least one ER stressmarker in said at least one biological sample higher than an expressionvalue of said at least one ER stress marker in said predeterminedstandard indicates that said treatment should be continued.

In other embodiments the at least one ER stress marker is bindingimmunoglobulin protein (BiP), phosphorylated α subunit of eukaryoticinitiation factor 2 (p-eIF2a), phosphorylated Inositol Requiring 1(p-IRE), phosphorylated PKR-like ER kinase (p-PERK) or C/EBP homologousprotein (CHOP) or any combination thereof. Various embodiments of thepresent disclosure relate to an ER stress marker which is bindingimmunoglobulin protein (BiP).

In some embodiments the expression level of said at least one ER stressmarker in step (a) is determined in at least two temporally separatedbiological samples of said patient. In other specific embodiments one ofsaid at least two biological samples is obtained before initiation ofsaid treatment. In further embodiments the at least two temporallyseparated biological samples are separated by a week, two, three or fourweeks, by a month, two, three or four months.

In various embodiments the method as herein defined further comprisesadministering the isolated peptide comprising the amino acid sequencedenoted by SEQ ID NO. 1 or a functional derivative thereof or apharmaceutically acceptable salt of said isolated peptide to saidpatient. In some embodiments the isolated peptide comprising the aminoacid sequence denoted by SEQ ID NO. 1 or a functional derivative thereofor a pharmaceutically acceptable salt of said isolated peptide isadministered at a dose of about 5 mg/m² to about 100 mg/m². In otherembodiments the isolated peptide comprising the amino acid sequencedenoted by SEQ ID NO. 1 or a functional derivative thereof or apharmaceutically acceptable salt of said isolated peptide isadministered at a frequency of once, twice or trice per week.

In further embodiments treatment as herein defined is with an isolatedpeptide consisting of the amino acid sequence denoted by SEQ ID NO. 1 orwith a pharmaceutically acceptable salt of said isolated peptide.

In various embodiments of the present disclosure cancer is selected fromthe group consisting of pancreatic cancer, ovarian cancer, spindle cellneoplasm of neural origin, spindle cell neoplasm, metastatic colorectalcancer, colon cancer, colorectal cancer, colon adenocarcinoma, rectalcancer, rectal adenocarcinoma, lung cancer, non-small cell lungcarcinoma, spinal cord neoplasm, breast cancer, skin cancer, renalcancer, multiple myeloma, thyroid cancer, prostate cancer,adenocarcinoma, head and neck cancer, gastrointestinal cancer, stomachcancer, cancer of the small intestine, hepatic carcinoma, liver cancerand malignancies of the female genital tract. In other specificembodiments cancer is selected from the group consisting of spindle cellneoplasm of neural origin, metastatic colorectal cancer, colon cancer,lung cancer, rectal cancer, pancreatic cancer and spinal cord neoplasm.In still further embodiments cancer cells in the patient are ST2positive cells.

In some embodiments the method according to the invention comprisescontacting at least one detecting agent specific for said at least oneER stress marker with said at least one biological sample or with anynucleic acid or protein product obtained therefrom. In variousembodiments the at least one detecting agent specific for said at leastone ER stress marker is an antibody or an antibody conjugated to adetectable moiety, wherein said antibody specifically recognizes andbinds said ER stress marker.

The present disclosure further provides a detecting agent specific foran ER stress marker for use in a method of predicting the response of acancer patient to treatment with an isolated peptide comprising theamino acid sequence denoted by SEQ ID NO. 1 or a functional derivativethereof or a pharmaceutically acceptable salt of said isolated peptide,said method comprising the steps of:

(a) determining the expression level of said ER stress marker with saiddetecting agent in at least one biological sample of said patient toobtain an expression value, wherein at least one of said biologicalsamples is obtained after the initiation of treatment;(b) determining if the expression value of said ER stress markerobtained in step (a) is higher or lower with respect to a predeterminedstandard expression value of said ER stress marker; wherein anexpression value of said ER stress marker obtained in (a) higher than anexpression value of said ER stress marker in a predetermined standardindicates that said patient is a responder to said treatment.

The present disclosure further provides a kit comprising:

(a) at least one detecting agent specific for determining the expressionvalue of at least one ER stress marker in a biological sample; andoptionally at least one of:

(b) predetermined standard expression values of said at least one ERstress marker determined for cancer patients before initiation oftreatment and upon administration of an isolated peptide comprising theamino acid sequence denoted by SEQ ID NO. 1 or any functional derivativethereof or a pharmaceutically acceptable salt of said isolated peptide;(c) at least one control sample.

In some embodiments the kit for determining the expression value of atleast one ER stress marker further comprising at least one reagent fordetermining the level of expression of at least one ER stress marker ina biological sample.

In other embodiments the kit for determining the expression value of atleast one ER stress marker further comprises:

(d) an isolated peptide comprising the amino acid sequence denoted bySEQ ID NO. 1 or a functional derivative thereof, or a pharmaceuticallyacceptable salt of said isolated peptide.

In further embodiments the kit for determining the expression value ofat least one ER stress marker further comprises instructions for use.

In various embodiments the kit as herein defined is for use inpredicting the response of a cancer patient to treatment with anisolated peptide comprising the amino acid sequence denoted by SEQ IDNO. 1 or a functional derivative thereof or a pharmaceuticallyacceptable salt of said isolated peptide.

The present disclosure further provides a combination therapy comprisingan anti-cancer agent and an isolated peptide comprising the amino acidsequence denoted by SEQ ID NO. 1 or a functional derivative thereof or apharmaceutically acceptable salt of said isolated peptide for use in amethod of treating cancer, wherein said anti-cancer agent isadministered at a dose lower than the standard of care dose of saidanti-cancer agent.

In some embodiments the combination therapy for use is wherein theadministered dose of said anti-cancer agent is lower than the standardof care dose of said anti-cancer agent by at least about 1%-50%, about5%-45%, about 10%-40%, about 15%-35% or about 20%-30%.

In other embodiments the combination therapy for use is wherein saidanti-cancer agent is a chemotherapeutic agent, a tyrosine kinaseinhibitor, an immunotherapy agent, a hormone agent, a biological agent,a differentiation factor, an anti-angiogenic factor, an anti-autophagyagent or an immune-stimulatory agent. In further embodiments thecombination therapy for use is wherein said anti-cancer agent is Taxol.

In still further embodiments the combination therapy for use is whereinsaid isolated peptide and said anti-cancer agent are administeredconcomitantly or consecutively.

In various embodiments of the aspect of combination therapy for usecancer is pancreatic cancer, ovarian cancer, spindle cell neoplasm ofneural origin, spindle cell neoplasm, metastatic colorectal cancer,colon cancer, colorectal cancer, colon adenocarcinoma, rectal cancer,rectal adenocarcinoma, lung cancer, non-small cell lung carcinoma,spinal cord neoplasm, breast cancer, skin cancer, renal cancer, multiplemyeloma, thyroid cancer, prostate cancer, adenocarcinoma, head and neckcancer, gastrointestinal cancer, stomach cancer, cancer of the smallintestine, hepatic carcinoma, liver cancer or malignancies of the femalegenital tract. In specific embodiments of the aspect of combinationtherapy for use cancer is ovarian cancer or pancreatic cancer. In stillfurther embodiments of the aspect of combination therapy for use cancercomprises ST2 positive cancer cells.

In some embodiments of the aspect of combination therapy for use theisolated peptide consists of the amino acid sequence denoted by SEQ IDNO. 1 or a pharmaceutically acceptable salt thereof.

In other embodiments of the aspect of combination therapy for use theisolated peptide or a pharmaceutically acceptable salt thereof isadministered at a dose of about 5 mg/m² to about 100 mg/m². In furtherembodiments of the aspect of combination therapy for use the isolatedpeptide or a pharmaceutically acceptable salt thereof is administered ata frequency of once, twice or trice per week.

By another one of its aspects the present disclosure provides atherapeutic kit comprising:

(a) an anti-cancer agent; and

(b) an isolated peptide comprising the amino acid sequence denoted bySEQ ID NO. 1 or a functional derivative thereof or a pharmaceuticallyacceptable salt of said isolated peptide.

In some embodiments the therapeutic kit is wherein said kit furthercomprises instructions for use.

In other embodiments the therapeutic kit is for use in a method oftreating cancer, wherein said anti-cancer agent is administered at adose lower than the standard of care dose of said anti-cancer agent.

By still a further aspect the present disclosure provides a method oftreatment of cancer in a patient in need thereof, comprisingadministering to said patient a therapeutically effective amount of anisolated peptide comprising the amino acid sequence denoted by SEQ IDNO. 1 a functional derivative thereof or a pharmaceutically acceptablesalt of said isolated peptide in combination with an anti-cancer agent,wherein said isolated peptide reduces the standard of care administereddose of said anti-cancer agent.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosedherein and to exemplify how it may be carried out in practice,embodiments will now be described, by way of non-limiting example only,with reference to the accompanying drawings, in which:

FIG. 1 is a summary of patients' randomization and assignments, showingpatient number and assignment to a specific cohort as well as the dosingused in the study.

FIG. 2A-FIG. 2B are micrographs of biopsy taken from a patientdesignated 002-006 suffering from spinal cord neoplasm. FIG. 2Adescribes binding immunoglobulin protein (BiP) staining of a biopsyobtained from the patient prior to receiving dTCApFs and FIG. 2Bdescribes BiP staining of a biopsy obtained after 11 months of treatmentwith dTCApFs. The antibody used for staining was an anti-BiP antibody(Abcam).

FIG. 3 is a graph showing the correlation between change in the serumlevel of the ER stress marker BiP and the dose of dTCApFs.

FIG. 4 is a graph showing the correlation between change in the serumlevel of the ER stress marker BiP and the change in tumor size.

FIG. 5 is a graph showing the number of days of participation in theclinical trial for patients according to their T1/ST2 expression.

FIG. 6 is a graph showing the correlation between change in the serumlevel of the ER stress marker BiP and the change in tumor size for ST2negative and ST2 positive populations.

FIG. 7 is a graph showing the serum concentrations of dTCApFs over timeby dose groups. Error bars represent SD.

FIG. 8 is a graph showing correlation between changes in tumor size andthe administered dose of dTCApFs.

FIG. 9A-FIG. 9D are immunocytochemistry images using an antibodydirected to β-cop of control OV90 cells (FIG. 9A), OV90 cells treatedwith the dTCApFs peptide as indicated (FIG. 9B), of control OV90 ST2knock-out (KO) cells (FIG. 9C) and of OV90 ST2 knock-out (KO) cellstreated with the dTCApFs peptide as indicated (FIG. 9D).

FIG. 10A-FIG. 10D are immunocytochemistry images using an antibodydirected to GRP78 BiP of control OV90 cells (FIG. 10A), OV90 cellstreated with the dTCApFs peptide as indicated (FIG. 10B), of controlOV90 ST2 knock-out (KO) cells (FIG. 10C) and of OV90 ST2 knock-out (KO)cells treated with the dTCApFs peptide as indicated (FIG. 10D).

FIG. 11A-FIG. 11B are bar graphs showing the serum CRT level indTCApFs-treated patients at the end of the treatment (FIG. 11A) and thechange in the CRT level in dTCApFs-treated patients (FIG. 11B).

FIG. 12 is a graph showing the activation of natural killer cells (NKcells) in the presence of dTCApFs.

FIG. 13A-FIG. 13B are bar graphs showing the level of BrdU incorporationin human ovarian cancer cells (FIG. 13A) and human pancreatic cancercells (FIG. 13B) in the presence of Taxol (2 nM or 4 nM), dTCApFs (25μg/ml) or a combination thereof (dTCApFs at 25 μg/ml and Taxol at 2 or 4nM).

FIG. 14 is a graph showing the change in tumor volume during theindicated period of time, in mice inoculated with human triple negativebreast cancer (hTNBC) cells and treated with dTCApFs (once a week, at 15mg/kg), doxorubicin (once a week, at 3 mg/kg) or with a combination ofdTCApFs and doxorubicin when doxorubicin and dTCApFs were administeredon the same day or when doxorubicin was administered 24 hours afterdTCApFs. “Control” represents treatment of mice with 5% mannitol.Abbreviations: Dox, doxorubicin; dTCApFs+Dox same day, a combination ofdTCApFs and doxorubicin administered on the same day; dTCApFs+Dox nextday, a combination of dTCApFs and doxorubicin where doxorubicin wasadministered 24 hours after administration of dTCApFs.

FIG. 15 is a graph showing survival rate of mice inoculated with hTNBCtumor and treated with Nerofe and doxorubicin during the indicatedperiod of time. Mice were treated with dTCApFs (once a week at 15mg/kg), doxorubicin (once a week at 3 mg/kg) or with a combination ofdTCApFs and doxorubicin when doxorubicin was administered on the sameday as dTCApFs or 24 hours after dTCApFs. “Control” represents treatmentof mice with 5% mannitol. Abbreviations: Dox, doxorubicin; dTCApFs+Doxsame day, a combination of dTCApFs and doxorubicin administered on thesame day; dTCApFs+Dox next day, a combination of dTCApFs and doxorubicinwhere doxorubicin was administered 24 after administration of dTCApFs.

FIG. 16A-FIG. 16B are micrographs showing fluorescence of KRAS in miceinoculated with MDA231 cells (hTNBC cells) that were not treated (FIG.16A) or treated with a combination of dTCApFs and doxorubicin (FIG.16B).

FIG. 17 is a graph showing the change in tumor volume during theindicated period of time, in mice inoculated with B16 cells and treatedwith dTCApFs (once a week at 15 mg/kg), doxorubicin (once a week at 3mg/kg) or with a combination of dTCApFs and doxorubicin when dTCApFs anddoxorubicin were administered on the same day. “Control” representstreatment of mice with 5% mannitol. Abbreviations: Dox, doxorubicin;dTCApFs+Dox same day, a combination of dTCApFs and doxorubicinadministered on the same day.

FIG. 18 is a graph showing the change in tumor size during the indicatedperiod of time, in mice inoculated with B16 cells (melanoma tumors) andtreated with anti-PDL1 antibodies (twice a week, at 20 mg/kg) alone orin combination with dTCApFs (three times a week, at 1 mg/kg). “Control”represents treatment of mice with 5% mannitol. Abbreviations: anti-PDL1ab, anti-PDL1 antibodies; anti-PDL1 ab+dTCApFs, anti-PDL1 antibodies incombination with dTCApFs.

FIG. 19A-FIG. 19B are photographs of a mice inoculated with a melanomatumor and treated with an a nti-PDL1 antibody in the presence of dTCApFs(FIG. 19A) or in its absence (FIG. 19B). Tumor area is indicated by anarrow.

FIG. 20A-FIG. 20D are fluorescence micrographs showing the presence ofNK cells and CD8 cells in tumor sections treated with a combination ofanti-PDL1 antibodies and dTCApFs (FIG. 20A and FIG. 20B, respectively)or with dTCApFs alone (FIG. 20C and FIG. 20D, respectively).

FIG. 21 is a graph showing the change in tumor size during the indicatedperiod of time, in mice inoculated with Panc02 cells and treated withdTCApFs (three times per week, at 1 mg/kg), anti-PDL1 antibodies (twicea week at 20 mg/kg) or with a combination of dTCApFs and anti-PDL1antibodies, when dTCApFs was administered three times per week at 1mg/kg and the anti-PDL1 antibodies were administered twice per week, at20 mg/kg. “Control” represents treatment of mice with 5% mannitol.Abbreviations: anti-PDL1 ab, anti-PDL1 antibodies; anti-PDL1 ab+dTCApFs,anti-PDL1 antibodies in combination with dTCApFs.

FIG. 22 is a graph showing the change in tumor size during the indicatedperiod of time, in mice inoculated with EMT6 cells and treated withdTCApFs (three times per week, at 1 mg/kg), anti-PDL1 antibodies (twicea week at 20 mg/kg) or with a combination of dTCApFs and anti-PDL1antibodies, when dTCApFs was administered three times per week at 1mg/kg and the anti-PDL1 antibodies were administered twice per week, at20 mg/kg. “Control” represents treatment of mice with 5% mannitol.Abbreviations: anti-PDL1 ab, anti-PDL1 antibodies; anti-PDL1 ab+dTCApFs,anti-PDL1 antibodies in combination with dTCApFs.

DETAILED DESCRIPTION OF EMBODIMENTS

The present disclosure is based on the surprising finding that treatmentwith the peptide termed herein dTCApFs (or Nerofe), which has the all Damino acid sequence of Trp Trp Thr Phe Phe Leu Pro Ser Thr Leu Trp GluArg Lys (denoted by SEQ ID NO: 1) increased endoplasmic reticulum (ER)stress in a variety of cancers, as evidenced from an increase in thelevel of expression of the ER stress marker binding immunoglobulinprotein (BiP). Remarkably, the observed increase in the ER stress markerBiP was found to correlate with an inhibition of tumor growth indTCApFs-treated patients, as demonstrated by the decrease in tumor sizeat the end of the treatment period.

Thus, inter alia, the present disclosure shows that an ER stress marker,for example BiP, may be used as a biomarker for assessing the effect oftreatment with the peptide dTCApFs in cancer patients.

Therefore in one of its aspects the present disclosure provides a methodfor predicting the response of a cancer patient to treatment with anisolated peptide comprising the amino acid sequence denoted by SEQ IDNO. 1 or a functional derivative thereof or a pharmaceuticallyacceptable salt of said isolated peptide, said method comprising thesteps of:

(a) determining the expression level of at least one endoplasmicreticulum (ER) stress marker in at least one biological sample of saidpatient to obtain an expression value, wherein at least one of saidbiological samples is obtained after the initiation of said treatment;(b) determining if the expression value of said at least one ER stressmarker obtained in step (a) is higher or lower with respect to apredetermined standard expression value of said at least one ER stressmarker;wherein an expression value of said at least one ER stress markerobtained in (a) higher than an expression value of said at least one ERstress marker in a predetermined standard indicates that said patient isa responder to said treatment.

In other words, the present disclosure provides a method fordetermining, at an early stage (e.g. after about a month from treatmentinitiation as exemplified below or at further time-points), whether thecancer patient responds to treatment (i.e., whether the treatment issuitable) and to determine further treatment options based on theseconclusions, e.g. whether it is advisable or not to proceed with thistype of therapy.

Adjusting suitable treatment protocols is highly valuable and clinicallydesired in view of the fact that a large number of treatment protocolsare often associated with undesired side effects, and moreover, may beunsuccessful. Thus, optimizing a treatment protocol at early stagesafter initiation of treatment and/or throughout or after a treatmentperiod may avoid inadequate treatments, reduce unnecessary side effectsand drug burden and improve the chance of success.

The term “prediction” or “predicting” as used herein with reference tothe response of a cancer patient to treatment refers to thedetermination or evaluation of the likelihood that a patient willrespond either favorably, namely will have a beneficial response, orunfavorably (namely will not experience a beneficial response) totreatment as herein defined. A patient with an overall beneficialresponse to treatment as herein defined is referred to as a “responder”while a patient that responds unfavorably to treatment is referred to asa “non-responder”.

The term “response” in the context of the present disclosure refers tothe patient's overall outcome as a result of treatment, which may beassessed using any clinical parameters known to a skilled practitionerin the field of the invention. Thus the term “responder” in the contextof treatment as herein defined refers to a patient experiencing anoverall improvement in at least one clinical parameter as compared to anuntreated subject diagnosed with the same condition (e.g., the sametype, stage, degree and/or classification of the cancer disease asherein defined), or as compared to the clinical parameters of the samesubject prior to treatment initiation (or at the first day thereof,prior to the first administration) or as compared to the clinicalparameters of a patient population for which an improvement in at leastone clinical parameter was not achieved (defined herein as“non-responders”).

The term “non-responder” to treatment refers to a patient notexperiencing an improvement in at least one of the clinical parameterand is diagnosed with the same conditions (e.g., the same type, stage,degree and/or classification of the pathology), or experiencing theclinical parameters of the same subject prior to treatment as hereindefined.

The meaning of an “improvement in clinical parameters” in the context ofcancer is well known in the art, and includes but is not limited toreduction in tumor size; inhibition, at least partially, of tumorgrowth; reduction in the number of tumors; decrease in acceptabledisease markers (namely any marker known in the art that is used fordiagnosis or monitoring of a disease); inhibition (at least partial) oftumor cell metastasis; enhancement of anti-tumor immune response;relief, at least partial, of one or more symptoms associated with thetumor; increase in the length of survival following treatment; decreasedmortality at a given point of time following treatment; etc. Thus insome embodiments a beneficial response is a decrease in tumor burdenwhich may be assessed according to the RECIST guideline (17).

As detailed above the present disclosure provides a method forpredicting the response of a cancer patient to treatment as hereindefined by first determining the expression level of at least oneendoplasmic reticulum (ER) stress marker in at least one biologicalsample of said patient to obtain an expression value, wherein at leastone of said biological samples is obtained after the initiation oftreatment. Then in step (b) it is determined whether the expressionvalue of said at least one ER stress marker obtained in step (a) ishigher or lower with respect to a predetermined standard expressionvalue of said at least one ER stress marker. According to the method ofthe invention, when an expression value of said at least one ER stressmarker obtained in (a) is higher than an expression value of said atleast one ER stress marker in a predetermined standard, said patient isdiagnosed as a “responder” to treatment.

It must be understood that expression values as defined below that arehigher or lower in comparison with a corresponding predeterminedstandard expression value (or a cut-off value) of a control samplepredict whether the patient is a “responder” or a “non-responder”.

Therefore the method comprises examining whether the expression value ofany one of the tested ER stress markers is within the range of theexpression value of a standard population or a cutoff value for suchpopulation or higher than the expression value of a standard populationor a cutoff value for such population. The expression value is referredto as “higher” than a corresponding predetermined standard expressionvalue wherein the expression value obtained for a certain ER stressmarker at a certain time point of treatment equals to or is higher byany one of about 1% to 99.9%, specifically, about 1% to about 5%, about5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about90% to 95%, about 95% to 99%, or about 99% to 99.9% or more than anexpression value obtained at a corresponding time point of treatment fora standard population (namely a predetermined standard expressionvalue).

Accordingly the term “lower” refers to any expression value below apredetermined standard expression value, for example by any one of about1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about95% to 99%, or about 99% to 99.9%.

In other words, the specific expression values of the tested samples arecompared to a predetermined cutoff or standard values. As used hereinthe term “comparing” denotes any examination of the expression leveland/or expression values obtained for samples tested according to theinvention in order to discover similarities or differences between atleast two different samples. It should be noted that comparing accordingto the present invention encompasses the possibility to use a computerbased approach.

The term “predetermined standard expression value” or “predeterminedstandard expression values” as herein defined refers to expressionlevels, optionally normalized to obtain expression values as definedbelow, of an ER stress marker in a population of responders to treatmentas herein defined, preferably at time points corresponding to the timepoint(s) at which the expression values according to the method of theinvention are obtained. For example but not limited to the expressionlevels obtained for the patients enrolled in the clinical trialdescribed herein below. These patients serve as exemplary predeterminedstandard responder population or predetermined standard expression valuefor the ER stress marker BiP in samples obtained before the treatmentand samples obtained at day 29 of treatment.

It should be noted that a predetermined standard expression value,sometimes referred to simply as “cutoff” herein, is a value that meetsthe requirements for both high diagnostic sensitivity (true positiverate) and high diagnostic specificity (true negative rate). It should benoted that the terms “sensitivity” and “specificity” are used hereinwith respect to the ability of one or more ER stress markers, tocorrectly classify a sample as belonging to a pre-established populationassociated with responsiveness to treatment as herein defined.

In certain alternative embodiments, a control sample (or a controlbiological sample) may be used (instead of, or in addition to,predetermined standard expression value). Accordingly, the expressionvalues of the ER stress marker are compared to the expression values inthe control sample. The control sample may be obtained from at least oneof a healthy subject, a subject suffering from the same pathologicdisorder and is not treated by the isolated peptide as herein defined,the treated patient prior to treatment, a subject that responds totreatment and a non-responder subject.

As indicated above the present disclosure is based on the surprisingfinding that treatment with the peptide dTCApFs (denoted by SEQ IDNO: 1) increased stress in the endoplasmic reticulum (ER) in tumorsobtained from a variety of cancer patients, in correlation with positivepatients' response to the treatment. The ER has an important role in thefolding and maturation of newly synthesized proteins. ER stress, asknown in the art, refers to the disruption of ER homeostasis that ismanifested by the accumulation of misfolded and unfolded proteins in theER. ER stress activates complex signaling networks, for example thenetwork referred to as the Unfolded Protein response (UPR), which actsto reduce ER stress and to restore homeostasis.

The unfolded protein response (UPR) is initiated by three ERtransmembrane proteins: Inositol Requiring 1 (IRE1), PKR-like ER kinase(PERK), and Activating Transcription Factor 6 (ATF6). During unstressedconditions, the ER chaperone, immunoglobin binding protein (BiP) bindsto the luminal domains of these master regulators keeping them inactive.Upon ER stress, BiP dissociates from these sensors resulting in theiractivation.

The activated UPR regulates downstream effectors with the followingthree distinct functions: adaptive response, feedback control, and cellfate regulation. The UPR adaptive response includes inter aliaupregulation of molecular chaperones and protein processing enzymes toincrease folding. Feedback control involves the negative regulation ofUPR activation as ER homeostasis is being re-established. Cell fateregulation by the UPR plays an important role in the pathogenesis of ERstress-related disorder. As known in the art, when the cell encountersER stress that the UPR can mitigate, the cell will survive. However,during unresolvable ER stress conditions, the UPR fails to reduce ERstress and restore homeostasis, promoting cell death.

Other pathways associated with ER stress are theendoplasmic-reticulum-associated protein degradation (ERAD) which is acellular pathway that targets misfolded proteins of the endoplasmicreticulum for ubiquitination and subsequent degradation by aprotein-degrading complex (namely the proteasome) and ER stress-mediatedapoptosis.

Thus, the term “endoplasmic reticulum stress marker” or “ER stressmarker” refers to any molecule associated with endoplasmic reticulumstress response, for example but not limited to any molecule associatedwith the signaling network referred to as the unfolded protein response(UPR) that acts to reduce ER stress and restore homeostasis, anymolecule associated with endoplasmic-reticulum-associated proteindegradation (ERAD) or any molecule associated with ER stress-mediatedapoptosis.

In some embodiments the ER stress marker as herein defined isglucose-regulated protein (GRP-78) also termed binding immunoglobulinprotein (BiP), Inositol Requiring 1 (IRE1), PKR-like ER kinase (PERK),the α subunit of eukaryotic initiation factor 2 (eIF2α), type II ERtransmembrane transcription factor (ATF6) or C/EBP homologous protein(CHOP).

As detailed above an increase in the level of expression of the bindingimmunoglobulin protein (BiP) ER marker was detected in patientsadministered with the peptide dTCApFs, in correlation with an inhibitionof tumor growth in these patients.

Therefore in various embodiments the methods, detecting agent specificfor an ER stress marker for use and kit according to the presentdisclosure are wherein said ER stress marker is binding immunoglobulinprotein (BiP). The term binding immunoglobulin protein (BiP) andglucose-regulated protein GRP78 are used interchangeably and refer tothe same ER stress marker.

The term “Binding immunoglobulin protein” (BiP), also known as 78 kDaglucose-regulated protein (GRP-78) or heat shock 70 kDa protein 5(HSPA5), refers to a protein that in humans is encoded by the HSPA5 geneand that acts as a molecular chaperone located in the lumen of theendoplasmic reticulum (ER). BiP binds newly synthesized proteins as theyare translocated into the ER, and maintains them in a state competentfor subsequent folding and oligomerization. BiP is also an essentialcomponent of the translocation machinery, and plays a role in transportacross the ER membrane of aberrant proteins destined for degradation bythe proteasome. BiP is an abundant protein under all growth conditions,but its synthesis is markedly induced under conditions that lead to theaccumulation of unfolded polypeptides in the ER. In specific embodimentsof the present disclosure BiP is human BiP, having the accessionUniProtKB number P11021.

Specifically, by another one of its aspects the present disclosureprovides a method for predicting the response of a cancer patient totreatment with an isolated peptide comprising the amino acid sequencedenoted by SEQ ID NO. 1 or a functional derivative thereof or apharmaceutically acceptable salt of said isolated peptide, said methodcomprising the steps of:

(a) determining the expression level of BiP in at least one biologicalsample of said patient to obtain an expression value, wherein at leastone of said biological samples is obtained after the initiation of saidtreatment;

(b) determining if the expression value of BiP obtained in step (a) ishigher or lower with respect to a predetermined standard expressionvalue of BiP;

wherein an expression value of BiP obtained in (a) higher than anexpression value of BiP in a predetermined standard indicates that saidpatient is a responder to said treatment.

In other specific embodiments the ER stress marker according to thepresent disclosure is Inositol Requiring 1 (IRE1). IRE1, a type I ERtransmembrane kinase, senses ER stress by its N-terminal luminal domain.Upon sensing the presence of unfolded or misfolded proteins, IRE1dimerizes and autophosphorylates to become active.

In other specific embodiments the ER stress marker according to thepresent disclosure is PKR-like ER kinase (PERK). PERK is also a type IER transmembrane kinase, which when activated by ER stress,oligomerizes, autophosphorylates and then directly phosphorylates Ser51on the α subunit of eukaryotic initiation factor 2 (eIF2α).

Thus in further specific embodiments the ER stress marker according tothe present disclosure is the α subunit of eukaryotic initiation factor2 (eIF2α).

In still further specific embodiments, the ER stress marker according tothe present disclosure is type II ER transmembrane transcription factor(ATF6). ATF6 has two isoforms, ATF6α and ATF6β.

In other specific embodiments the ER stress marker according to thepresent disclosure is the ER stress-mediated apoptosis such as C/EBPhomologous protein (CHOP).

In still further specific embodiment the ER stress marker as hereindefined is a phosphorylated protein, for example p-eIF2α, p-IRE1 orp-PERK.

Therefore in the above and other embodiments the ER stress markeraccording to the present disclosure is binding immunoglobulin protein(BiP), phosphorylated α subunit of eukaryotic initiation factor 2(p-eIF2α), phosphorylated Inositol Requiring 1 (p-IRE), phosphorylatedPKR-like ER kinase (p-PERK) or C/EBP homologous protein (CHOP) or anycombination thereof.

In some embodiments the method, detecting agent specific for an ERstress marker for use or kit according to the present disclosureindicate that said treatment should be continued. In other words, insome embodiments of the method, detecting agent specific for an ERstress marker for use or kit according to the present disclosure anexpression value of said at least one ER stress marker in said at leastone biological sample higher than an expression value of said at leastone ER stress marker in said predetermined standard indicates that saidtreatment should be continued.

In some embodiments a patient is a responder to treatment in accordancewith the present disclosure when the patient experiences at least one ofcancer regression, progression-free survival, disease-free survival,complete response or partial response.

Alternatively, in case the expression value of said at least one ERstress marker in said at least one biological sample of a patient islower than the expression value of the same ER stress marker in apredetermined standard, treatment should be arrested.

As noted above, the methods of the present disclosure are based ondetermining the expression level of at least one ER stress marker inbiological samples. The terms “level of expression” or “expressionlevel” are used interchangeably and generally refer to a numericalrepresentation of the amount (quantity) of a polypeptide (protein) orpolynucleotide which encodes an amino acid product or protein in abiological sample. “Expression” generally refers to the process by whichgene-encoded information is converted into the structures present andoperating in the cell and may be evaluated via measurement of thequantity of the polypeptide (protein) or polynucleotide which encodesthereof.

Determination of the expression level of at least one ER stress markermay also be based on the expression level of fragments of thepolypeptide (protein) or the polynucleotide encoding thereof, or on theexpression level of any post-translationally modified protein (e.g.phosphorylated protein) or fragments thereof.

Determining the expression level of an endoplasmic reticulum stressmarker (e.g. BiP) or of any polypeptide fragment or derivative thereofor of any nucleic acid encoding thereof may be performed by any methodknown in the art, using a detecting agent specific for the endoplasmicreticulum stress marker being examined. For example, determining theexpression level of an ER stress marker may be performed using ELISA,immunoassay, immunofluorescence, immunohistochemistry,immunoprecipitation, northern blot, western blot, PCR, immuno-PCR, orsurface plasmon resonance.

In particular, determining the expression level of the ER stress markerBiP may be performed as described below using an ELISA-based method orusing labelled antibodies (e.g. labelled antibodies directed to BiP).Determining the expression level of an ER stress marker (e.g. BiP) mayalso be based on determining the expression level of a nucleic acid(e.g. mRNA) that encodes for BiP protein or by determining theexpression level of any polypeptide fragment or derivative of BiP in abiological sample.

In certain and specific embodiments, the step of determining the levelof expression to obtain an expression value by the method of theinvention further comprises an additional and optional step ofnormalization. According to this embodiment, in addition todetermination of the level of expression of the at least one ER stressmarker as herein defined, the level of expression of at least onesuitable control reference gene (e.g., housekeeping genes) is beingdetermined in the same sample. According to such embodiment, theexpression level of the at least one stress marker of the inventionobtained in step (a) is normalized according to the expression level ofsaid at least one reference control gene obtained in the additionaloptional step in said test sample, thereby obtaining a normalizedexpression value. Optionally, similar normalization is performed also inat least one control sample or a representing standard when applicable(namely the predetermined standard as herein defined).

Thus the term “expression value” refers to the result of a calculationthat uses as an input the “level of expression” or “expression level”obtained experimentally and by normalizing the “level of expression” or“expression level” by at least one normalization step as detailedherein, the calculated value termed herein “expression value” isobtained.

More specifically, as used herein, “normalized values” or “expressionvalues” are the quotient of raw expression values of ER stress markers,divided by the expression value of a control reference gene from thesame sample, such as a stably-expressed housekeeping control gene. Thedivision of the raw expression level of an ER stress marker by thecontrol reference gene raw expression level yields a quotient or ameasure which is essentially free from any technical failures orinaccuracies and constitutes a normalized expression value of saidmarker gene. This normalized expression value may then be compared withnormalized cutoff values, i.e., cutoff values calculated from normalizedexpression values. In certain embodiments, the control reference genemay be a gene that maintains stable in all samples analyzed in themicroarray analysis.

In specific embodiments the method and kit as herein defined comprisecontacting at least one detecting agent specific for said at least oneER stress marker with said at least one biological sample or with anynucleic acid or protein product obtained therefrom.

As indicated below the determination of the ER stress marker BiP wasbased on the use of antibodies directed to BiP. Therefore in someembodiments of the method, detecting agent specific for an ER stressmarker for use and kit according to the present disclosure the detectingagent specific for the (at least one) ER stress marker is an antibody oran antibody conjugated to a detectable moiety, wherein said antibodyspecifically recognizes and binds said ER stress marker.

By the term “detecting agent specific for an ER stress marker” as hereindefined it is meant any agent specific for an ER stress marker that maybe used for quantifying the level of the relevant ER stress markerexpression in a sample (for example but not limited to a nucleic acidagent specific for detecting an mRNA encoding the relevant ER stressmarker or a specific antibody that binds and recognizes the relevant ERstress marker or a fragment thereof).

Still further, it must be understood that any of the detecting agentspecific for an ER stress marker or reagents used by the kits and in anystep of the methods of the invention are non-naturally occurringproducts or compounds, As such, none of the detecting molecules of theinvention are directed to naturally occurring compounds or products.

In some embodiments the ER stress marker is BiP and the term “detectingagent specific for BiP” means any agent specific for BiP that may beused for quantifying the level of BiP expression in a sample (e.g. anucleic acid agent specific for detecting an mRNA coding for BiP or aspecific antibody that binds and recognizes BiP or fragment thereof).

The term “antibody” is used herein in its broadest sense and encompassesbut is not limited to a single chain antibody, a monoclonal antibody, abi-specific antibody, a chimeric antibody, a synthetic antibody, apolyclonal antibody, a humanized antibody, a fully human antibody, oractive fragments or homologues thereof. In the above and otherembodiments the antibody as herein defined is a non-naturally occurringantibody. An antibody conjugated to a detectable moiety refers to anyantibody conjugated to a detectable moiety so as to have a tag which isdetectable by fluorescence, chemiluminescence and the like (alsoreferred to herein as a “labelled antibody”).

The term “contacting” means to bring, put, incubate or mix together. Assuch, a first item is contacted with a second item when the two itemsare brought or put together, e.g., by touching them to each other orcombining them. In the context of the present invention, the term“contacting” includes all measures or steps which allow interactionbetween the at least one of the detection molecules for the ER stressmarkers (and optionally one suitable control reference gene) and thenucleic acid or amino acid molecules of the tested sample(s).

Thus by another one of its aspects the present disclosure provides adetecting agent specific for an ER stress marker for use in a method(performed in vitro) of predicting the response of a cancer patient totreatment with an isolated peptide comprising the amino acid sequencedenoted by SEQ ID NO. 1 or a functional derivative thereof or apharmaceutically acceptable salt of said isolated peptide, said methodcomprising the steps of:

(a) determining the expression level of said ER stress marker with saiddetecting agent in at least one biological sample of said patient toobtain an expression value, wherein at least one of said biologicalsamples is obtained after the initiation of treatment;(b) determining if the expression value of said ER stress markerobtained in step (a) is higher or lower with respect to a predeterminedstandard expression value of said ER stress marker;

wherein an expression value of said ER stress marker obtained in (a)higher than an expression value of said ER stress marker in apredetermined standard indicates that said patient is a responder tosaid treatment.

The method of the present invention further relates to repeateddetermination of the patient's response to treatment as herein defined,at various time points during treatment, namely in “temporallyseparated” biological samples of the patient, thereby monitoring thecontinued response of the patient. When the expression value of the atleast one ER stress marker obtained in step (a) of the method as hereindefined for each one of the temporally separated biological samples ishigher than the expression value of the same at least one ER stressmarker in a predetermined standard obtained for a correspondingpopulation (responders) at corresponding time point(s) along treatment,treatment may be continued.

For example the difference in the ER stress marker levels, e.g., BiPlevel, may be calculated by comparing the expression levels of the ERstress marker between two biological samples obtained from the samepatient. The first biological sample is a control biological sampleobtained from the patient prior to treatment or at the first day oftreatment (before the first administration) or the first biologicalsample is a predetermined standard expression level of the relevant ERstress marker, obtained from patients having the same condition andclinical status prior to treatment. The second biological sample is abiological sample of said patient obtained after the initiation oftreatment. The above procedure may be repeated.

For example and as exemplified below, the second biological sample maybe obtained about four weeks (e.g. on day 29) after initiation oftreatment and the first biological sample (the control biologicalsample) is obtained prior to treatment or at the first day of saidtreatment before the first administration. Specifically, when the ERstress marker is BiP, the difference is BiP expression levels ismeasured as follows: [(BiP level at day 29)−(BiP level at day1)]*100/(BiP level at day 1)

Therefore in specific embodiments the at least one biological sample instep (a) of the method as herein defined is obtained about four weeksafter the initiation of said treatment.

In various embodiments, the method, detecting agent specific for an ERstress marker for use or kit according to the present disclosure iswherein the expression level of said at least one ER stress marker instep (a) is determined in at least two temporally separated biologicalsamples of said patient.

The term “temporally separated” in the context of biological samples asherein defined refers to biological samples obtained at different timepoints, for example but not limited to prior to treatment and at day 29of treatment.

In other embodiments the method, detecting agent specific for an ERstress marker for use or kit according to the present disclosure arewherein one of said at least two biological samples is obtained beforeinitiation of said treatment, where “initiation of treatment” or“treatment initiation” should be taken to mean the administration of thefirst dose of the isolated peptide as herein defined. By the term“before initiation of said treatment” it is meant before the firstadministration of the isolated peptide. In some embodiments the term“before initiation of said treatment” refers to the first day oftreatment before the first administration is made.

In other specific embodiments the method, detecting agent specific foran ER stress marker for use or kit according to the present disclosureis wherein said at least two temporally separated biological samples areseparated by a week, two, three or four weeks, by a month, two, three orfour months. In further embodiments the method, detecting agent specificfor an ER stress marker for use or kit according to the presentdisclosure is wherein said at least two temporally separated biologicalsamples are separated by more than four months.

The term “biological sample” is used herein in its broadest sense andrefers to samples obtained from a mammal subject. Biological samples maybe obtained from mammals (including humans) and encompass fluids, solidsand tissues. In some embodiments the biological sample is blood, plasma,serum, lymph fluid, urine, a tissue sample, a biopsy sample or a celllysate.

As detailed above the biological sample is obtained after the initiationof treatment as herein defined, or before (prior to) the initiation ofsaid treatment. Biological samples may be obtained by any method knownin the art by a skilled physician.

As described in the Examples below, the effect of dTCApFs was alsoexamined on human natural killer (NK) cells. As shown in FIG. 12, anincrease in expression of the receptors CD335 and CD337 was induced bydTCApFs. Natural killer cells provide a rapid response to viral-infectedcells and respond to tumor formation. Without wishing to be bound bytheory, the activation of the receptors CD335 and CD337 by dTCApFs isassociated with increasing ER stress in these cells. Therefore in someembodiments measurement of the ER stress may be performed in patients'cells, e.g. NK cells.

Furthermore the term “at least one” with reference to a biologicalsample refers to one, two, three, four, five, six, seven, eight, nine ormore biological samples. Mutatis mutandis the term “at least one” in thecontext of ER stress markers refers to one, two, three, four, five, six,seven, eight, nine or more ER stress markers.

According to the results shown in Example 1 below, a clear difference inthe level of BiP was observed in a biopsy obtained from a spinal cordneoplasm patient before treatment was initiated (FIG. 2A) as compared tothe level of BiP observed in a biopsy obtained from the same patientafter 11 month of treatment (FIG. 2B). The above results demonstratethat the dTCApFs peptide increased ER stress in tumors obtained from aspinal cord neoplasm cancer patient.

In addition, the results shown in Example 2 below demonstrate that anincrease in the level of BiP correlated with complete inhibition oftumor growth in patients having different cancers types that weretreated with the peptide dTCApFs under the conditions specified herein.

As used herein to describe the present disclosure, “cancer” or “tumor”relate equivalently to a hyperplasia of a tissue or organ. If the tissueis a part of the lymphatic or immune systems, malignant cells mayinclude non-solid tumors of circulating cells. Malignancies of othertissues or organs may produce solid tumors. In general, the methods andcompositions of the present disclosure may be used in the treatment ofnon-solid and solid tumors.

In some embodiments cancer is selected from the group consisting ofpancreatic cancer, ovarian cancer, spindle cell neoplasm of neuralorigin, spindle cell neoplasm, metastatic colorectal cancer, coloncancer, colorectal cancer, colon adenocarcinoma, rectal cancer, rectaladenocarcinoma, lung cancer, non-small cell lung carcinoma, spinal cordneoplasm, breast cancer, skin cancer, renal cancer, multiple myeloma,thyroid cancer, prostate cancer, adenocarcinoma, head and neck cancer,gastrointestinal cancer, stomach cancer, cancer of the small intestine,hepatic carcinoma, liver cancer and malignancies of the female genitaltract.

In further embodiments of the present disclosure cancer is selected fromthe group consisting of spindle cell neoplasm of neural origin,metastatic colorectal cancer, colon cancer, lung cancer, rectal cancer,pancreatic cancer and spinal cord neoplasm.

In the clinical study described below a correlation was found betweenthe anti-tumour activity of dTCApFs and the T1/ST2 expression status inpatients' tumour cells. A direct correlation was found betweenT1/ST2-positivity, tumour size changes and induction of ER stress. Thesefindings are consistent with the preclinical studies described below inExample 4 showing that incubation of ST2 gene knockout OV-90 cells withdTCApFs did not result in ER stress. Without wishing to be bound bytheory these observations suggest that the T1/ST2 receptor may be abiomarker for selecting T1/ST2 positive patients who are more likely topositively respond to dTCApFs.

Therefore in still further embodiments the methods of the presentdisclosure further comprises determining the ST2 receptor status of thecells in biological sample(s) obtained from the patient under dTCApFstreatment. As detailed below, the changes from baseline in the levels ofsoluble T1/ST2 receptor and peripheral blood mononuclear cell (PBMC)T1/ST2 receptor expression are also monitored in pretreatment and/oron-treatment tumor tissue samples obtained from the patient undergoingtreatment with dTCApFs.

Without wishing to be bound by theory, expression of the ST2 receptor oncancer cells facilitates dTCApFs entry into the cells. Therefore in someembodiments of the method, detecting agent specific for an ER stressmarker for use or kit as herein defined the cancer cells in patients areST2 positive cells.

The term “ST2 receptor” or T1/ST2 receptor (also referred to as “T1/ST2”and “ST2/T1”) as herein defined refers to a member of the IL-IRsuperfamily, which possesses three extracellular immunoglobulin domainsand an intracellular TIR domain. T1/ST2 has been indicated as beinginvolved in cardiovascular disease. The term “ST2 positive cells” asherein defined refers to cells for which the presence of the ST2receptor on the cells is identified by any method known in the art forexample but not limited to the method described below.

In further embodiments the method or detecting agent specific for an ERstress marker for use according to the present disclosure is wherein themethod further comprises administering the isolated peptide comprisingthe amino acid sequence denoted by SEQ ID NO. 1 or a functionalderivative thereof or a pharmaceutically acceptable salt of saidisolated peptide to the patient.

Therefore by still a further aspect the present disclosure provides amethod for predicting the response of a cancer patient to treatment withan isolated peptide comprising the amino acid sequence denoted by SEQ IDNO. 1 or a functional derivative thereof or a pharmaceuticallyacceptable salt of said isolated peptide, said method comprising thesteps of:

(a) administering said isolated peptide comprising the amino acidsequence denoted by SEQ ID NO. 1 or a functional derivative thereof orsaid pharmaceutically acceptable salt of said isolated peptide to thepatient;

(b) determining the expression level of at least one endoplasmicreticulum (ER) stress marker in at least one biological sample of saidpatient to obtain an expression value, wherein at least one of saidbiological samples is obtained after the initiation of said treatment;(c) determining if the expression value of said at least one ER stressmarker obtained in step (b) is higher or lower with respect to apredetermined standard expression value of said at least one ER stressmarker;

wherein an expression value of said at least one ER stress markerobtained in (a) higher than an expression value of said at least one ERstress marker in a predetermined standard indicates that said patient isa responder to said treatment.

The isolated peptide comprising the amino acid sequence denoted by SEQID NO. 1 or a functional derivative thereof or a pharmaceuticallyacceptable salt of said isolated peptide may be administered by anyroute of administration known to a person skilled in the art, forexample intravenously (iv).

The isolated peptide as herein defined may be administered at an“effective amount” such that necessary to achieve the desiredtherapeutic result. The “effective amount” is determined by the severityof the disease in conjunction with the therapeutic objectives, the routeof administration and the patient's general condition (age, sex, weightand other considerations known to the attending physician).

As detailed below, an on-going clinical trial is being performed by theinventors, in accordance with which the cancer type, dose,administration frequency, treatment length, administration and otherparameters were determined. As detailed below, the dosing regimen was 6,12, 24, 48 or 96 mg/m² (for example as shown in FIG. 1).

In various embodiments the isolated peptide comprising the amino acidsequence denoted by SEQ ID NO. 1 or a functional derivative thereof or apharmaceutically acceptable salt of said isolated peptide isadministered at a dose of about 5 mg/m² to about 100 mg/m², 90 mg/m², 80mg/m², 70 mg/m², 60 mg/m² or about 10 mg/m² to about 50 mg/m².

In specific embodiments the method or detecting agent specific for an ERstress marker for use as herein defined is wherein said isolated peptidecomprising the amino acid sequence denoted by SEQ ID NO. 1 or afunctional derivative thereof or said pharmaceutically acceptable saltof said isolated peptide is administered at a dose of about 5 mg/m² toabout 100 mg/m².

In further specific embodiments the isolated peptide comprising theamino acid sequence denoted by SEQ ID NO. 1 or a functional derivativethereof or a pharmaceutically acceptable salt of said isolated peptideis administered at a dose of about 6, 12, 24, 48 or 96 mg/m² dTCApFs.

In still further specific embodiments the method, detecting agentspecific for an ER stress marker for use or kit as herein defined iswherein said isolated peptide comprising the amino acid sequence denotedby SEQ ID NO. 1 or a functional derivative thereof or a pharmaceuticallyacceptable salt of said isolated peptide is administered at a frequencyof once, twice or trice per week.

In yet further specific embodiments the isolated peptide comprising theamino acid sequence denoted by SEQ ID NO. 1 or a functional derivativethereof or a pharmaceutically acceptable salt of said isolated peptideis administered at a frequency of 3 times per week.

As exemplified below treatment with dTCApFs (at 6, 12, 24, 48 and 96mg/m², 3 times/week, in consecutive 28-day cycles) in locally advancedor metastatic solid tumors was safe and well-tolerated, with a dosedependent, linear PK. dTCApFs suppressed antigenic factors, inducedanti-cancer cytokines production, and ER stress, which probably led tothe clinical outcome observed in some of the patients. Positive T1/ST2staining could serve as a predictive marker for response to dTCApFs.

Therefore the present invention further provides a method of treatmentof cancer in a patient in need thereof, comprising administering to saidpatient a therapeutically effective amount of an isolated peptidecomprising the amino acid sequence denoted by SEQ ID NO. 1, a functionalderivative thereof or a pharmaceutically acceptable salt of saidisolated peptide. In some embodiments the therapeutically effectiveamount of the isolated peptide is about 6, 12, 24, 48, or 96 mg/m². Inyet further embodiments the isolated peptide is administered three timesper week for a period of at least about four consecutive weeks. In stillfurther specific embodiments the isolated peptide is the peptide termedherein dTCApFs consisting of the amino acid sequence denoted by SEQ IDNO: 1.

In other specific embodiments the therapeutically effective amount ofthe isolated peptide is about 24, 48, or 96 mg/m² or higher. In furtherspecific embodiments the therapeutically effective amount is about 24mg/m² (or higher), for a period of at least about four weeks and thepatient is further administered with the isolated peptide at a highertherapeutically effective amount of about 48, 96 mg/m² or higher.

The terms “treat”, “treating”, “treatment” as used herein meanameliorating, alleviating or eliminating one or more clinicalparameters, symptoms or indications of disease activity in a patienthaving cancer. The clinical parameters associated with cancer as knownin the art and as detailed above are for example tumor size, tumorgrowth, number of tumors, disease markers, tumor cell metastasis etc. By“patient” it is meant any mammal for which administration of theisolated peptide as herein defined, or any pharmaceutical composition ofthe invention is desired, namely patient afflicted with cancer as hereindefined, in particular human patients.

Monitoring the treatment as herein defined may be performed by any meansknown in the art for monitoring cancer patient's response to treatment,for example according to the RECIST guideline (17).

As detailed below the present disclosure concerns inter alia resultsassociated with an on-going clinical trial performed with the peptidedTCApFs, having the all D amino acid sequence of Trp Trp Thr Phe Phe LeuPro Ser Thr Leu Trp Glu Arg Lys (as denoted by SEQ ID NO: 1).

The term “isolated peptide” as herein defined encompasses an isolatedpeptide comprising the amino acid sequence denoted by SEQ ID NO. 1(namely the amino acid sequence Trp Trp Thr Phe Phe Leu Pro Ser Thr LeuTrp Glu Arg Lys in an all D conformation), termed herein “dTCApFs” or“Nerofe” and functional derivatives of the amino acid sequence denotedby SEQ ID NO. 1 or pharmaceutically acceptable salts of said isolatedpeptide.

Any pharmaceutically acceptable salt of the isolated peptide as hereindefined are encompassed by the present disclosure, in particular theacetate salt of the peptide.

In some embodiment the isolated peptide consists of the amino acidsequence denoted by SEQ ID NO. 1, having the all D amino acid sequenceof Trp Trp Thr Phe Phe Leu Pro Ser Thr Leu Trp Glu Arg Lys. In specificembodiments the isolated peptide according to the present disclosure isa pharmaceutically acceptable salt of the amino acid sequence denoted bySEQ ID NO. 1, for example the acetate salt thereof.

In other words in various embodiments the method, detecting agentspecific for an ER stress marker for use or kit according to theinvention is wherein said treatment is with an isolated peptideconsisting of the amino acid sequence denoted by SEQ ID NO. 1 or with apharmaceutically acceptable salt of said isolated peptide.

In other specific embodiments the present disclosure provides a methodfor predicting the response of a cancer patient to treatment with anisolated peptide consisting of the amino acid sequence denoted by SEQ IDNO. 1 or with a pharmaceutically acceptable salt of said isolatedpeptide, said method comprising the steps of:

(a) determining the expression level of BiP in at least one biologicalsample of said patient to obtain an expression value, wherein at leastone of said biological samples is obtained after the initiation of saidtreatment;

(b) determining if the expression value of BiP obtained in step (a) ishigher or lower with respect to a predetermined standard expressionvalue of BiP;

wherein an expression value of BiP obtained in (a) higher than anexpression value of BiP in a predetermined standard indicates that saidpatient is a responder to said treatment

In other words, the present disclosure provides methods, detecting agentspecific for an ER stress marker for use, composition and kit forpredicting a cancer patient's response to treatment with an isolatedpeptide consisting of the amino acid sequence denoted by SEQ ID NO. 1 orwith a pharmaceutically acceptable salt of said isolated peptide.

The term “peptide” as herein defined refers to a molecular chain ofamino acid residues, which, if required, can be modified at each one ofits amino acid residues, for example by manosylation, glycosylation,amidation (for example C-terminal amides), carboxylation orphosphorylation. The peptide may be obtained synthetically, throughgenetic engineering methods, expression in a host cell, or through anyother suitable means. Methods for producing peptides are well known inthe art.

The term “isolated” refers to molecules, such as amino acid sequences orpeptides that are removed from their natural environment, isolated orseparated.

The term “amino acid” as used herein, refers to naturally occurring andsynthetic amino acid residues, as well as amino acid analogs and aminoacid mimetics that function in a manner similar to the naturallyoccurring amino acids. Naturally occurring amino acids are those encodedby the genetic code, as well as those amino acids that are latermodified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.

The term amino acid encompasses L-amino acids and D-amino acids, whichare mirror images of L-amino acids, where the chirality at carbon alphahas been inverted. D-amino acids are highly resistant to proteasemediated degradation and have a low immunogenic response.

The terms “amino acid sequence” or “peptide sequence” also relate to theorder in which amino acid residues, connected by peptide bonds, lie inthe chain in peptides and proteins. The sequence is generally reportedfrom the N-terminal end containing free amino group to the C-terminalend containing free carboxyl group.

By the term “comprising” it is meant that the isolated peptide inaccordance with the present disclosure includes the peptide denoted bySEQ ID NO: 1, but may also include additional amino acid residues at theN-terminus or at the C-terminus of the peptide or at both termini.

As indicated above, the present disclosure also encompasses isolatedpeptides comprising derivatives of the peptide having the amino acidsequence denoted by SEQ ID NO. 1.

By the term “derivative” or “derivatives” it is meant to includepeptides, which comprise the amino acid sequence denoted by SEQ ID NO:1, but differ in one or more amino acids in their overall sequence,namely, which have deletions, substitutions (e.g. replacement of atleast one amino acid by another amino acid), inversions or additionswithin the overall sequence of SEQ ID NO: 1. This term also encompassesthe replacement of at least one amino acid residue in the overallsequence by its respective L amino acid residue.

In particular embodiments the present disclosure relates to a functionalderivative of the amino acid sequence denoted by SEQ ID NO. 1, whereinsaid functional derivative has at least 70%, 75%, 80%, 85%, 90%, morepreferably 95%, in particular 99% identity to the amino acid sequencedenoted by SEQ ID NO: 1.

Amino acid “substitutions” are the result of replacing one amino acidwith another amino acid having similar structural and/or chemicalproperties, i.e., conservative amino acid replacements. Amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, each of thefollowing eight groups contains amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M).

It is appreciated that these peptide derivatives must not alter thebiological activity of the original peptide. The term “functional” meansto denote that the modified peptide (namely the derivative) retains abiological activity qualitatively similar to that of the unmodifiedpeptide. The biological activity of the derivative may be determined asherein described, namely by monitoring the effect of said derivativeupon administration to an animal model, as known in the art.

In some embodiments the isolated peptide as herein defined or apharmaceutically acceptable salt thereof is comprised in apharmaceutical composition.

The term “pharmaceutical compositions” as herein defined refers to theisolated peptide comprising the amino acid sequence denoted by SEQ IDNO. 1 or a functional derivative thereof, or a pharmaceuticallyacceptable salt of said isolated peptide and optionally at least onepharmaceutically acceptable excipient or carrier as known in the art. Asused herein “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic composition is contemplated.

Pharmaceutical compositions used to treat subjects in need thereofaccording to the present disclosure optionally also comprise a bufferingagent, an agent who adjusts the osmolarity thereof, and optionally, oneor more pharmaceutically acceptable additives as known in the art.

Pharmaceutical compositions used to treat subjects in need thereofaccording to the invention, which may conveniently be presented in unitdosage form, may be prepared according to conventional techniques wellknown in the pharmaceutical industry, for example as detailed in theExamples below.

It should be understood that in addition to the ingredients particularlymentioned herein, the compositions according to the present disclosuremay also include other agents conventional in the art having regard tothe type of formulation in question.

Still further the present disclosure provides a kit comprising:

(a) at least one detecting agent specific for determining the expressionvalue of at least one ER stress marker in a biological sample; andoptionally at least one of:

(b) predetermined standard expression values of said at least one ERstress marker determined for cancer patients before initiation oftreatment and upon administration of an isolated peptide comprising theamino acid sequence denoted by SEQ ID NO. 1 or any functional derivativethereof or a pharmaceutically acceptable salt of said isolated peptide;(c) at least one control sample.

By the term “detecting agent specific for determining the expressionvalue” of at least one ER stress marker it is meant any detecting agentspecific for an ER stress marker as herein defined and optionally anadditional detecting agent specific for determining the level ofexpression of at least one suitable control reference gene, as definedabove.

The control sample may comprise a biological sample or anypolypeptide/nucleic acid derived therefrom.

In specific embodiments the kit according to the present disclosurefurther comprises at least one reagent for determining the level ofexpression of at least one ER stress marker in a biological sample. Anyreagents known in the art for such purpose are encompassed, for examplebut not limited to secondary antibodies dyes and fluorescent agents.

In further embodiment the kit according to the present disclosurefurther comprises:

(d) an isolated peptide comprising the amino acid sequence denoted bySEQ ID NO. 1 or a functional derivative thereof, or a pharmaceuticallyacceptable salt of said isolated peptide.

In other embodiments the kit according to the present disclosure furthercomprises instructions for use. Such instructions may comprise at leastone of: instructions for carrying out the determination of theexpression value of at least one ER stress marker in a biologicalsample; and instructions for comparing the expression values of at leastone ER stress marker in a biological sample to the predeterminedstandard expression values of said at least one ER stress marker.

In still further embodiments the kit according to the present disclosureis wherein said at least one ER stress marker is BiP.

In other embodiments the kit according to the present disclosure is foruse in predicting the response of a cancer patient to treatment with anisolated peptide comprising the amino acid sequence denoted by SEQ IDNO. 1 or a functional derivative thereof or a pharmaceuticallyacceptable salt of said isolated peptide.

As exemplified below higher abundance of ST2 receptors on cellsfacilitated the entry of the dTCApFs peptide to cells. Without wishingto be bound by theory, this may enable increasing ER stress in cancercells of treated patients, and may enable lowering the doses of theadministered isolated peptide or any additional agent administeredtherewith.

Most anti-cancer agents are strong medicines that have a fairly narrowdose range for safety and effectiveness reasons. Taking too little of anagent will not treat the cancer well and taking too much may causelife-threatening side effects. Chemotherapy is known for the adverseeffects associated therewith. Common side effects are, among others,fatigue, pain, mouth and throat sores, diarrhea, nausea and vomiting.Ways for limiting or reducing the doses of administered chemotherapeuticagents are therefore desirable.

As indicated below, the beneficial therapeutic effect of the dTCApFspeptide on cancer cells and the understanding of its unique associationwith ER stress served as a basis for a further aspect of the presentinvention, according to which dTCApFs is combined with anotheranti-cancer therapeutic agent. Without wishing to be bound by theory, asa consequence of the effect of dTCApFs on ER stress, dTCApFs may allow asignificant reduction in the administered amount of an additionalanti-cancer agent, and thereby indirectly reduce the associated sideeffects thereof while maintaining the anti-tumor effect of the drug.

A study in which dTCApFs was administered in combination with anadditional anti-cancer therapeutic agent, for example Taxol is describedbelow. Surprisingly, while each one of the agents, namely dTCApFs andTaxol, had only a marginal effect on cell viability, combining dTCApFswith Taxol resulted in a synergistic effect and substantial reduction incell viability. Therefore, a combination of dTCApFs with an anti-canceragent allows the reduction of the standard of care administered dose ofthe anti-cancer agent during cancer therapy.

Therefore by still another one of its aspects the present disclosureprovides a combination therapy comprising an anti-cancer agent and anisolated peptide comprising the amino acid sequence denoted by SEQ IDNO. 1 or a functional derivative thereof or a pharmaceuticallyacceptable salt of said isolated peptide for use in a method of treatingcancer, wherein said anti-cancer agent is administered at a dose lowerthan the standard of care dose of said anti-cancer agent.

The term “combination therapy” as used herein refers to concomitant(simultaneous) or consecutive administration of two or more agents,namely an isolated peptide comprising the amino acid sequence denoted bySEQ ID NO. 1 or a functional derivative thereof and an anti-canceragent. For example, concurrent administration can mean one dosage formcontaining the two or more agents or administration of a mixture of thetwo or more agents whereas consecutive administration means separatedosage forms administered to the patient at different time points andmay be even by different routes of administration.

Thus in some embodiments of the present disclosure the isolated peptideof the invention and said anti-cancer agent are administeredconcomitantly or consecutively.

Thus the term “standard of care dose of said anti-cancer agent” as usedherein refers to a dose recommended by a skilled physician for treatmentof a certain type of cancer in a cancer patient based on considerationssuch as the anti-cancer agent(s) to be administered, the patient's age,gender, weight and relevant clinical parameters associated with thedisease and the general health condition of the patient. While someanti-cancer agents are determined according to the patient body weightin kilograms, for some anti-cancer agent doses are determined based onbody surface area (BSA), which doctors calculate using height andweight. BSA is expressed in meters squared (m²). Dosages for childrenand adults differ, even after BSA is taken into account.

As indicated below, combining the peptide dTCApFs with Taxol resulted ina synergistic effect and substantial reduction in cell viability.Therefore, a combination therapy of the dTCApFs peptide with ananti-cancer agent may allow the reduction of the standard of careadministered dose of the anti-cancer agent during cancer therapy.

In some embodiments of the present disclosure the combination therapyfor use according to the present disclosure is wherein the administereddose of said anti-cancer agent is lower than the standard of care doseof said anti-cancer agent by at least about 1%-50%, about 5%-45%, about10%-40%, about 15%-35% or about 20%-30%.

The term “anti-cancer agent” also known as “anticancer drug” or“antineoplastic drug” is used in its broader sense and encompasses anydrug or agent that is effective in the treatment of malignant orcancerous disease. There are several classes of anticancer drugs, interalia alkylating agents (e.g. Cyclophosphamide), antimetabolites (e.g.5FU), natural products, immunotherapeutic agent, hormones andinhibitors.

In some embodiments the anti-cancer agent according to the presentdisclosure is a chemotherapeutic agent, a tyrosine kinase inhibitor, animmunotherapy agent (e.g. an antibody, an antibody fragment or amonoclonal antibody that down-regulates inhibitory immune receptors), ahormone agent, a biological agent, a differentiation factor, ananti-angiogenic factor, an anti-autophagy agent or an immune-stimulatoryagent.

A “chemotherapeutic agent” as known in the art is a drug that targetscells at different phases of the process of forming new cells. Examplesof chemotherapeutic agent include but are not limited to alkylatingagents, antimetabolites (e.g. 5-FU), anti-tumour antibiotics,topoisomerase inhibitors, mitotic inhibitors (e.g. Paclitaxel or Taxol)or corticosteroids, to name but few.

The term “tyrosine kinase inhibitor” as known in the art refers to adrug that inhibits tyrosine kinases. Tyrosine kinases are enzymesresponsible for the activation of many proteins by signal transductioncascades. The proteins are activated by adding a phosphate group to theprotein (phosphorylation), a step that tyrosine kinase inhibitorsinhibit.

The terms “an immunotherapy agent” or “immune-stimulatory agent” in thecontext of the present disclosure refers to cancer immunotherapy, whichattempts to stimulate the immune system to destroy tumours.

The term “biological agent” in the context of cancer treatment as knownin the art (sometimes referred to as “immune therapy”) involves the useof living organisms, substances derived from living organisms, orlaboratory-produced versions of such substances to treat disease. Somebiological therapies for cancer use vaccines or bacteria to stimulatethe body's immune system to act against cancer cells. Biologicaltherapies that interfere with specific molecules involved in tumourgrowth and progression are also referred to as targeted therapies.

The term “anti-angiogenic factor” as known in the art refers to an agentthat interferes with angiogenesis, the process of creation of new bloodvessels. Anti-angiogenesis agents are types of targeted therapy that usedrugs or other substances to stop tumours from making the new bloodvessels they need to keep growing.

The term “anti-autophagy agent” as known in the art refers to a drugthat interferes with the process autophagy, namely the regulated,destructive mechanism of the cell that disassembles unnecessary ordysfunctional components (e.g. bleomycin, doxorubicin).

In further embodiments the anti-cancer agent according to the presentdisclosure is a chemotherapeutic agent, a tyrosine kinase inhibitor, animmunotherapy agent, a hormone agent, a biological agent, adifferentiation factor, an anti-angiogenic factor, an anti-autophagyagent or an immune-stimulatory agent

In specific embodiments the anti-cancer agent is Taxol.

In some embodiments the present disclosure provides a combinationtherapy comprising an anti-cancer agent and an isolated peptideconsisting of the amino acid sequence denoted by SEQ ID NO. 1 or apharmaceutically acceptable salt of said isolated peptide for use in amethod of treating cancer, wherein said anti-cancer agent isadministered at a dose lower than the standard of care dose of saidanti-cancer agent.

In further specific embodiments the present disclosure provides acombination therapy comprising taxol and an isolated peptide consistingof the amino acid sequence denoted by SEQ ID NO. 1 or a pharmaceuticallyacceptable salt of said isolated peptide for use in a method of treatingcancer, wherein taxol is administered at a dose lower than the standardof care dose of said anti-cancer agent.

In some embodiments the isolated peptide or the pharmaceuticallyacceptable salt thereof is administered at a dose of about 5 mg/m² toabout 100 mg/m².

In further embodiments the isolated peptide or a pharmaceuticallyacceptable salt thereof is administered at a frequency of once, twice ortrice per week. In specific embodiments the isolated peptide or apharmaceutically acceptable salt thereof is administered at a frequencyof three times per week.

In still further embodiments the isolated peptide, the anti-canceragent, or any pharmaceutically acceptable salt thereof, together orseparately are comprised in a pharmaceutical composition.

In some embodiments the combination therapy for use according to thepresent disclosure is wherein said cancer is pancreatic cancer, ovariancancer, spindle cell neoplasm of neural origin, spindle cell neoplasm,metastatic colorectal cancer, colon cancer, colorectal cancer, colonadenocarcinoma, rectal cancer, rectal adenocarcinoma, lung cancer,non-small cell lung carcinoma, spinal cord neoplasm, breast cancer, skincancer, renal cancer, multiple myeloma, thyroid cancer, prostate cancer,adenocarcinoma, head and neck cancer, gastrointestinal cancer, stomachcancer, cancer of the small intestine, hepatic carcinoma, liver canceror malignancies of the female genital tract.

In other embodiments, the combination therapy for use according to thepresent disclosure is wherein said cancer is ovarian cancer orpancreatic cancer. In further embodiments, the combination therapy foruse according to the present disclosure is wherein said cancer is breastcancer, preferably wherein said breast cancer is triple negative breastcancer (TNBC).

In further embodiments the combination therapy for use according to thepresent disclosure is wherein said cancer comprises ST2 positive cancercells.

In still further embodiments the combination therapy for use accordingto the present disclosure is wherein said isolated peptide consists ofthe amino acid sequence denoted by SEQ ID NO. 1 or a pharmaceuticallyacceptable salt thereof.

In further embodiments the combination therapy for use according to thepresent disclosure is wherein said isolated peptide or apharmaceutically acceptable salt thereof is administered at a dose ofabout 5 mg/m² to about 100 mg/m².

In still further embodiments the combination therapy for use accordingto the present disclosure is wherein said isolated peptide or apharmaceutically acceptable salt thereof is administered at a frequencyof once, twice or trice per week.

The present disclosure further provides a therapeutic kit comprising:

(a) an anti-cancer agent; and

(b) an isolated peptide comprising the amino acid sequence denoted bySEQ ID NO. 1 or a functional derivative thereof or a pharmaceuticallyacceptable salt of said isolated peptide.

In some embodiments the therapeutic kit as herein defined furthercomprises instructions for use.

In some embodiments the therapeutic kit as herein disclosed is for usein a method of treating cancer, wherein said anti-cancer agent isadministered at a dose lower than the standard of care dose of saidanti-cancer agent.

The present disclosure further provides a method of treatment of cancerin a patient in need thereof, comprising administering to said patient atherapeutically effective amount of an isolated peptide comprising theamino acid sequence denoted by SEQ ID NO. 1 a functional derivativethereof or a pharmaceutically acceptable salt of said isolated peptidein combination with an anti-cancer agent, wherein said isolated peptidereduces the standard of care administered dose of said anti-cancer age.

The term “about” as used herein indicates values that may deviate up to1%, more specifically 5%, more specifically 10%, more specifically 15%,and in some cases up to 20% higher or lower than the value referred to,the deviation range including integer values, and, if applicable,non-integer values as well, constituting a continuous range.

Disclosed and described, it is to be understood that this invention isnot limited to the particular examples, methods steps, and compositionsdisclosed herein as such methods steps and compositions may varysomewhat. It is also to be understood that the terminology used hereinis used for the purpose of describing particular embodiments only andnot intended to be limiting since the scope of the present inventionwill be limited only by the appended claims and equivalents thereof.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”. This termencompasses the terms “consisting of” and “consisting essentially of”.The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the claimed composition or method.Throughout this specification and the Examples and claims which follow,unless the context requires otherwise, the word “comprise”, andvariations such as “comprises” and “comprising”, will be understood toimply the inclusion of a stated integer or step or group of integers orsteps but not the exclusion of any other integer or step or group ofintegers or steps.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise.

Throughout this specification and the Examples and claims which follow,unless the context requires otherwise, the word “comprise”, andvariations such as “comprises” and “comprising”, will be understood toimply the inclusion of a stated integer or step or group of integers orsteps but not the exclusion of any other integer or step or group ofintegers or steps.

The following examples are representative of techniques employed by theinventors in carrying out aspects of the present invention. It should beappreciated that while these techniques are exemplary of preferredembodiments for the practice of the invention, those of skill in theart, in light of the present disclosure, will recognize that numerousmodifications can be made without departing from the spirit and intendedscope of the invention.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe claimed invention in any way.

Standard molecular biology protocols which are known in the art and notspecifically described herein are generally followed as in Sambrook &Russell, 2001.

Experimental Procedures

Biopsy

Biopsies were obtained from patients by procedures well known in theart, by a skilled physician.

BiP Staining of Tissues Obtained by Biopsy

Solutions and Reagents

-   -   Xylene (Sigma #534056);    -   Ethanol, anhydrous denatured, histological grade (100% Solufix        #E003 and 95% Sigma #32294);    -   Deionized water (dH₂O);    -   Hematoxylin Gill2 (Sigma #GHS216);    -   Wash Buffer: 1×TBS/0.1% Tween-20 (1×TBST): For preparation of 1        L, 50 ml 20×TBS (Amresco #J640) were added to 950 ml dH₂O, 1 ml        Tween-20 (Amresco #J640) was added and the buffer was mixed;    -   Antibody Diluent: SignalStain® Antibody Diluent #8112;    -   Antigen Unmasking:        -   Citrate: 10 mM Sodium Citrate Buffer: For the preparation of            1 L, 2.94 gr sodium citrate trisodium salt dihydrate            (C₆H₅Na₃O₇.2H₂O) were added to 1 L dH₂O. pH was adjusted to            6.0.        -   TE: 10 mM Tris/1 mM EDTA, pH 9.0: For the preparation of 1            L, 1.21 gr Trizma® base (C₄H₁₁NO₃) and 0.372 g EDTA were            added to 1 L dH₂O.    -   3% Hydrogen Peroxide: For the preparation of 100 mL solution, 10        ml 30% H₂O₂ (Sigma #216763) were added to 90 ml dH₂O;    -   Blocking Solution: Background Buster (Innovex #NB306);    -   Primary antibody: Anti BiP antibody for Immunohistochemistry        (Anti-BIP ab21685, abcam);    -   Biotinylated secondary antibody: SignalStain® Boost        Immunohistochemistry (IHC) Detection Reagent (HRP, Rabbit)        #8114;    -   DAB Reagent (Sigma #D6190);    -   Eukitt Mounting Media (Sigma #03989);        Deparaffinization/Rehydration

Slides were not allowed to dry at any time during this procedure.Sections of biopsy tissues were prepared as known in the art and weredeparaffinized/hydrated as follows: sections were incubated in threewashes of xylene for 5 minutes each, further incubated in two washes of100% ethanol for 10 minutes each, next incubated in two washes of 95%ethanol for 10 minutes each and finally sections were washed twice indH₂O for 5 minutes each.

Antigen Unmasking

For Citrate, Slides were boiled in 10 mM sodium citrate buffer pH 6.0,then maintained at a sub-boiling temperature for 10 minutes. Slides werecooled on the bench top for 30 minutes. For Tris EDTA (TE), slides wereboiled in 10 mM TE/1 mM EDTA, pH 9.0 then maintained at a sub-boilingtemperature for 18 minutes. Slides were cooled on the bench for 30minutes.

Staining

Sections were washed in dH₂O three times for 5 minutes each, incubatedin 3% hydrogen peroxide for 10 minutes, washed in dH₂O twice for 5minutes each and washed in wash buffer for 5 minutes. Then, each one ofthe sections was blocked with 100-400 μl blocking solution for 30minutes at room temperature. The Blocking solution was removed and100-400 μl primary antibody (namely anti-BiP) diluted 1:500 in antibodydiluent was added to each one of the sections. The treated sections wereincubated overnight at 4° C. Next, the antibody solution was removed andthe sections were washed in wash buffer three times for 5 minutes each.Biotinylated secondary antibody (100-400 μl) was added to each one ofthe sections and the treated sections were incubated 30 minutes at roomtemperature in the presence of the secondary antibody. Then, thesecondary antibody solution was removed and the sections were washedthree times with wash buffer for 5 minutes each. Next, the reagent3,3′-diaminobenzidine (DAB, 100-400 μl) was added to each one of thesections and the staining was monitored closely. As soon as the sectionsdeveloped, slides were immersed in dH₂O. Sections were optionallycounterstained in hematoxylin per manufacturer's instructions and thenwashed in dH₂O two times for 5 minutes each.

Dehydration of Sections

Sections were incubated in 95% ethanol two times for 10 seconds each,the incubation was repeated in 100% ethanol, incubating sections twotimes for 10 seconds each. The incubation was then repeated in xylene,incubating sections two times for 10 seconds each and then thecoverslips were mounted for analysis.

Preparation of the dTCApFs Peptide and Composition Comprising Thereof

The peptide dTCApFs or NEROFE™ (both terms are used hereininterchangeably and refer to the same peptide as indicated above) is a14 amino acid residues long peptide, in which all of the amino acidresidues are at their D configuration, having the amino acid sequence ofTrp Trp Thr Phe Phe Leu Pro Ser Thr Leu Trp Glu Arg Lys (orWWTFFLPSTLWERK in a single letter code, as denoted by SEQ ID NO: 1).

The peptide was synthesized as follows. dTCApFs Acetate, the final drugproduct for use in clinical studies, was manufactured, packaged, tested,labeled and released under Good Manufacturing Practices (GMP) by NextarLtd., Israel.

dTCApFs powder for solution for injection was supplied either as alyophilized 5 mL vial containing 15 mg (at a concentration of 7.5 mg/mL)or 10 mL vial containing 80 mg (at a concentration of 40 mg/mL) of theactive substance with 4.8% mannitol, for reconstitution to a finalvolume of 2 mL with water for injection (WFI), per vial.

The reconstituted 2 mL vial of dTCApFs powder for solution for injectionmust thereafter be diluted to a final volume of 100 or 250 mL in aqueousDextrose 5% for infusion. dTCApFs was supplied by Nextar Ltd for thedesignated clinical site for use in a phase one clinical study.

The drug product is a white sterile, non-pyrogenic lyophilized cake forsingle reconstitution in water for injection. Following reconstitution,it has the appearance of clear, colorless solution. Vials are type Iclear injection glass 5 mL or 10 mL vials, stoppered with 20 mmlyophilization type rubber stoppers, with a 20 mm aluminum flip-off cap.Each ten vials are secondary packaged in a white, labeled outer box.Vial and secondary packaging box clinical study GCP standard labeling isperformed under controlled conditions by Nextar Ltd.

On-Going Clinical Trial Outline

Patients

The study included adults patients (≥18 years) with pathologicallyconfirmed locally advanced and/or metastatic solid malignancies, whofailed or could not tolerate previous standard therapy. Key inclusioncriteria included evaluable/measurable disease and Eastern CooperativeOncology Group (ECOG) performance status (PS)≤1. Patients with livercancer/hepatic metastases were eligible if liver function met certaincriteria, and patients with brain metastases were eligible if radiationtherapy was completed ≥4 weeks prior to enrollment and the patientreceived ≤4 mg/day of dexamethasone. Key exclusion criteria includedreceiving anti-cancer treatment 14 days prior to initiation of studydrug and life expectancy of <16 weeks. Patients characteristics aresummarized in Table 1 below.

TABLE 1 Patients demographics and baseline characteristics dTCApFs dose6 mg/m² 12 mg/m² 24 mg/m² 48 mg/m² 96 mg/m² n = 3 n = 3 n = 3 n = 3 n =3 Age, years Median (range) 63 (62-77) 61 (58-62) 65 (57-67) 72 (51-94)64 (55-77) Mean (SE) 68 (5) 67 (4) 67 (2) 72 (8) 64 (9) Gender,male/female, n/n 3/0 2/1 1/2 2/1 3/2 Tumor, type, n Colorectal 3 2 0 2 1Pancreatic 0 0 1 0 4 Other^(a) 0 1 2 1 0 Prior therapies, n Chemotherapy3 4 4 1 3 Radiotherapy 1 2 1 1 0 Surgery 2 2 1 1 2 Treatment withbiological agents 0 0 1 0 0 Treatment with small molecules 0 0 0 1 0such as tyrosine kinase inhibitors ^(a)Includes neoplasms in the smallintestine, lung, liver, and spinal cord.Study Design

The present clinical study is a formal open label phase I doseescalation study. The primary objective was to determine the maximumtolerated dose (MTD) and safety profile of dTCApFs. Assessments includeddrug exposure, adverse events (AEs; graded according to the CommonTerminology Criteria for Adverse Events (CTCAE), and characterization ofdose-limiting toxicities (DLTs). Other objectives included assessment ofserum levels of angiogenic factors after dTCApFs administration,pharmacokinetics (PK) and pharmacodynamics (PD) analyses, as well asassessment of receptor staining and tumor response.

The dose escalation study followed a traditional “3+3” scheme andincluded doses of 6, 12, 24, 48, and 96 mg/m² intravenous (i.v.)dTCApFs, 3 times/week in consecutive 28-day cycles. Patients'assignments are presented in FIG. 1. In all 3-patient cohorts, therewere 2-4 weeks between the first dose for the first and second patients,and ≥1 week for the third patient. New dose levels started after followup of ≥28 days for 3 patients at the previous level. MTD was defined asthe highest dose level at which ≥1 of 3 subjects experience a DLT duringtheir first cycle of treatment. Patients who did not complete theirfirst cycle of treatment for reasons unrelated to AEs were replaced. Inaddition, PK parameters, including area under the curve (AUC), maximalplasma concentration (Cmax), and plasma half-life (t½) were determined.PK parameters were estimated using non-compartmental models.

In other words patients were administered i.v. with dTCApFs at 6 mg/m²,3 times per week, as long as their disease was not progressing. If adose of 12 mg/m² was proven to be safe and the disease was progressingthen the patient was administered with 12 mg/m². For example, patientnumber 1 in FIG. 1 was administered with two cycles of treatment (at 6and 12 mg/m² dTCApFs). Patient number 4 in FIG. 1 received three cyclesof treatment (at 12, 24 and 48 mg/m² dTCApFs).

Clinical activity of dTCApFs was assessed every 8 weeks by physicalexamination, computed tomography (CT), or magnetic resonance imaging(MRI) techniques (for evaluable disease only), using RECIST v1.1; and,where appropriate, informative tumor markers every cycle. This study wasapproved by the institutional review board of Rabin Medical Center, andthe Ministry of Health, Israel and conducted at the Davidoff Center,Rabin Medical Center in accordance with the Declaration of Helsinki. Allpatients signed an informed consent before enrollment. The study wasregistered at ClinicalTrials.gov (NCT01690741).

Administration

dTCApFs powder for solution for injection is supplied as a lyophilized 5mL vial containing either 15 mg (7.5 mg/mL) or 10 mL vial containing 80mg (40 mg/mL) of the active substance with 4.8% mannitol, forreconstitution to a final volume of 2 mL with water for injection pervial as described above. The reconstituted 2 mL vial of dTCApFs powderfor solution for injection must thereafter be diluted to a final volume100 mL in aqueous Dextrose 5% for infusion for dose levels up to andincluding 48 mg/m2. For dose levels above 48 mg/m2, the final dilutionvolume was 250 mL. dTCApFs for injection was administered intravenously(iv) during 60 minutes.

Pharmacokinetic Analyses

PK parameters, including AUC(0-24), Cmax, Cmin, Tmax and t_(1/2) areestimated using non-compartmental models. Comparisons across dose levelsare made to assess proportionality. A summary of the PK parameters isprovided in Table 2 below.

TABLE 2 Pharmacokinetics of dTCApFs on the first day of cycles 1 and 2(each cycle was 28 days). dTCApFs dose 6 mg/m² 12 mg/m² 24 mg/m² 48mg/m² 96 mg/m² n = 3 n = 4 n = 4 n = 4 n = 3 Cycle 1, Day 1 AUC₀, ng ·h/mL 3813 12,905 49,630 79,935 206,742 C_(max), ng/mL 1209 6048 14,60918,267 32,964 T_(1/2), h 2.3 2.1 3.2 4.9 6.0 Cycle 2, Day 1 AUC₀, ng ·h/mL 9719 11,452 57,069 100,093 294,682 C_(max), ng/mL 1536 6048 14,60922,113 32,016 T_(1/2), h 2.8 2.0 3.7 4.6 8.5Pharmacodynamic Analyses

Changes from baseline in the levels of circulating cytokine and solubleT1/ST2 receptor and peripheral blood mononuclear cell (PBMC) T1/ST2receptor expression are presented for interpretation and correlated withPK and antitumor activity analyses. If pretreatment and/or on-treatmenttumor tissue samples are obtained, results of T1/ST2 receptors assaysare presented for clinical interpretation.

Biomarker Analysis

Blood samples were collected from patients and placed on ice for 10minutes on a regular basis as described below. Serum was collected bycentrifuging at 3000 rpm for 10 minutes at 4° C., was kept in a separatevial at ≤−20° C., and shipped to Immune System Key Ltd at −20° C., wherethey were thawed, aliquoted, and stored at ≤−20° C. Repeated freeze-thawcycles were avoided.

Immunohistochemistry (IHC) staining was performed for T1/ST2 receptorusing a full length anti-ST2 antibody (GenMed, Plymouth, Minn.). Serumlevels of various factors were measured with enzyme-linked immunosorbentassay (ELISA). Additional factors that were measured included: Vascularendothelial growth factor (VEGF), Vascular endothelial growth factor D(VEGF-D), epidermal growth factor (EGF), angiopoietin-1, fibroblastgrowth factor 1 (FGF-1), fibroblast growth factor 2 (FGF-2),platelet-derived growth factor AA (PDGF-AA), platelet-derived growthfactor BB (PDGF-BB), transforming growth factor β1 (TGF-β1) (all usingELISA kits by R&D systems, Abingdon, UK); granulocyte-macrophagecolony-stimulating factor (GM-CSF), interleukin 2 (IL-2), interleukin12p70 (IL-12p70), interleukin 21 (IL-21) and tumor necrosis factor α(TNF-α) (Millipore, Billerica, Mass.); and glucose regulated protein 78(GRP78)/BiP (Enzo, New York, N.Y.). A summary of serum levels of variousangiogenic factors and cytokines measured in patients undergoingtreatment with the peptide is presented in Table 3 below.

TABLE 3 Mean change in serum levels of angiogenetic factors andcytokines with dTCApFs administration dTCApFs dose 6 mg/m² 12 Mg/m² 24Mg/m² 48 Mg/m² 96 Mg/m² n = 3 n = 3 n = 3 n = 3 n = 5 Change in serumlevel from pre- to post- treatment with dTCApFs, % Angiogenic factorsAngioeitin-1 +960 −80 −77 −50 +70 FGF-1 +120 −62 −20 −27 +457 FGF-2 +199−74 −34 −13 +44 PDGF-AA +1379 −92 −79 −73 +57 PDGF-BB +2271 −95 −82 −78+185 VEGF-A +265 −47 −62 −72 −2 TGF-β1 +18 −80 −59 −20 No data VEGF-D+117 −40 −54 −63 +3 Cytokines GM-CSF +2173 −97 +11 +5613 +974 IL12-p70+469 −76 +83 +477 +332 Il-2 No data −100 No data +242 +577 Il-21 +100−61 +84 +1326 +29 TNF-α +4 −5 +31 +74 +97 FGF, fibroblast growth factor;GM-CSF, granulocyte-macrophage colony-stimulating factor; IL,interleukin, PDGF, platelet-derived growth factor; TGF, transforminggrowth factor; VEGF, vascular endothelial growth factor; TNF, tumornecrosis factor.Immunogenicity

Changes from baseline in the levels of circulating anti-dTCApFsantibodies are presented for interpretation.

Antitumor Activity Analyses

Subjects with evaluable or measurable disease are assessed according tothe response evaluation criteria in solid tumors (RECIST) version 1.1every 2 cycles, where a cycle is defined as 4 weeks of treatment withthree administrations per week. Tumor lesion measurements and changesfrom baseline are summarized by cycle and dose cohort. A summary of theadverse events by dTCApFs dose group is provided in Table 4 below.

TABLE 4 Summary of adverse event by dTCApFs dose group dTCApFs dose 6mg/m² 12 mg/m² 24 mg/m² 48 mg/m² 96 mg/m² n = 3 n = 3 n = 3 n = 3 n = 5Grade 1 Blood disorders Anemia 3 0 0 0 0 Increased INR 0 0 0 1 0 GIdisorders Abdominal pain 0 1 2 0 0 Bowel obstruction 1 0 0 0 0 Diarrhea0 2 0 0 2 GI hemorrhage 1 0 0 0 0 Vomiting 2 0 0 0 1 Genera disordersDehydration 0 0 0 0 1 Fatigue 0 1 0 1 0 Hypertension 3 1 1 0 1 Nervoussystem disorders Neuropathy 0 1 1 0 0 Grade 2 Pain Pain, leg 0 2 0 0 0Pain, upper back 0 0 0 0 1 Respiratory system disorders Cough 0 1 0 0 0Skin disorders Pruritus 0 0 0 0 1 Urticaria 0 0 0 0 2 Hepatic andurinary disorders ALT increase 0 0 0 0 1 AST increase 0 0 0 0 1Bilirubin increase 0 0 0 1 1 Liver dysfunction 0 0 0 0 1 Urinary tractinfection 0 1 0 0 0 Grade 3 Blood disorders Increased INR 0 0 0 1 0General disorders Hypertension 2 1 1 0 2 Hepatic and urinary disordersBilirubin increase 0 0 0 1 1 GI disorders Bowel obstruction 1 0 0 0 0Diarrhea 0 1 0 0 1 GI hemorrhage 1 0 0 0 0 Grade 4 GI disorders Vomiting0 0 0 0 1 ALT, alanine transaminase; AST, aspartate aminotransferase;GI, gastrointestinal; INR, international normalized ratio.

Objective tumor response rates (complete response and partial response),duration of objective tumor response, time to objective tumor response,and progression-free survival are presented. Time-to-event estimates andsurvival curves are generated using the Kaplan-Meier method and Coxmodel with calculated crude Hazard ratio and calculated adjusted Hazardratio (adjusted for confounder variables). Subjects with informativetumor marker assessments (eg, CA125 or PSA) undergo appropriateassessments every cycle. Tumor marker parameters, when evaluable, aresummarized by cycle and dose cohort. An exploratory evaluation of therelationship between PK, PD and clinical effects of dTCApFs isperformed. A summary of the progression-free survival (PFS) of patientsenrolled in the study is presented in Table 5 below.

TABLE 5 PFS on the last regimen before enrolling the study and ondTCApFs. Greyed rows represent patients who experienced PFS on dTCApFswhich was comparable or exceeded that of their last regimen preenrollment. PFS on the last regimen pre Patient no. enrollment, days PFSon dTCApFs, days 1 480 53 2 134 25 3 110 170 4 0 330 5 52 51 6 384 110 754 90 8 80 52 9 375 60 10 1800 14 11 41 52 12 42 50 13 96 42 14 365 4015 1 80 16 105 45 17 564 41 PFS, progression-free survivalStatistical Analysis

Descriptive statistics were used for all analyses and were performedwith SAS® version 9.1 (SAS Institute Inc., Cary, N.C.). Regressionanalysis was used to study 2-way correlation between tumor change permonth, administered doses of dTCApF, and levels of the ER-stressbiomarker (BiP). The statistical significance of the correlation wasvalidated using F-statistics.

Determination of the GRP78/Bip Marker Blood Level by ELISA

Determination of the plasma level of BiP in patients undergoingtreatment by the dTCApFs peptide was performed as follows.

Samples Collection

Blood was collected from patients treated with dTCApFs in lavender topvacutainer tubes, then placed on ice for 10 minutes. Then the vacutainerwas centrifuged at 3000 rpm for 10 minutes at 4° C. The plasma fractionwas collected onto a separate vial and kept at ≤−20° C. until shipped tothe R&D department with World Courier at −20° C. When received, serumsamples were thawed, aliquot of 56 μl were made in 0.5 ml vials andstored at ≤−20° C. Repeated freeze-thaw cycles were avoided. Blood wascollected on days 1, 15 and 29 of the first cycle and on day 29 of thesubsequent cycles.

Determination of Blood Levels of GRP78/BIP

Determination of blood levels of GRP78/BIP was performed using the kitGRP/BIP ELISA kit (cat #ADI-900-214 Enzo. Aliquots of plasma sampleswere thawed at 4° C. and centrifuged at 10,000 G for 6 minutes at 4° C.The samples were then diluted 1:5 with Tris buffered saline containingBSA and detergents (kit assay) buffer. The samples were then loaded induplicates onto the provided 96 well plate, which were pre-coated withdonkey anti-sheep IgG. Calibration samples and blank samples were alsoloaded onto the plate, and then the antibody directed to BiP (yellow)was added to all of the wells, except the blank ones. The ELISA platewas sealed and incubated at room temperature (RT) with shaking at 750rounds per minute (RPM) for 1 hour.

After the above incubation period the plate was not washed and BiPconjugate (blue) was added to all of the wells except the blank ones.The plate was incubated with shaking for 1 additional hour (RT).

After the additional incubation period, the wells content was aspiratedand the wells were washed using an automated wash (Bio-plex pro II Washstation) by adding 300 μl Tris buffered saline containing detergents(the kit's wash buffer) to every well. The washing procedure wasrepeated three more times for a total of four washes. After the finalwash, the wells were aspirated and the plate was tapped firmly upsidedown on a lint free paper towel to remove any residual wash buffer.

3,3′,5,5′-Tetramethylbenzidine (TMB) solution was added into each well,and the plate was sealed and incubated for 19 minutes at RT in the darkwith shaking. Stop solution was then added into each well, delta OD wasread at 450 nm/570 nm. Blank wells values were subtracted from allresults. Calibration curve was created and BiP ng/ml values werecalculated accordingly and multiplied with dilution factor (×5).

Calculating the Difference in Plasma BiP Expression Level

The difference in the plasma expression level of BiP was determined bycalculating the difference between BiP expression level measured in theplasma of a patient treated with dTCApFs on day 29 of treatment and BiPexpression level measured in the plasma of a patient treated withdTCApFs on day 1 of treatment (prior to the first administration of thedTCApFs peptide) and by dividing the result by the above BiP expressionlevel measured on day 1 of treatment (normalizing), namely as follows:[(BiP level at day 29)−(BiP level at day 1)]*100/(BiP level at day 1).

Determination of Calreticulin (CRT) Blood Level

Serum CRT levels were determined using the kit Elisa CRT kit (human)(OKEH01054, Aviva system Biology). Patients' samples from days 1 and 15or 29 of cycle 1 (C1D1, C1D15 and C1D29) were thawed and centrifuged at10 minutes at 10,000 G and loaded onto the plate according to themanufacturer's protocol.

Determination of Tumor Size

Tumor size was evaluated at the medical site according to the ResponseEvaluation Criteria in Solid Tumors (RECIST) guidelines (for example byperforming a computerized tomography scan (CT)). The tumor size on thelast day of the trial (namely after the last administration of thepeptide) was compared to the tumor size on the first day of the trial(prior to the first administration of the peptide), in percentage.

Evaluation of the ST2 Status in Cancer Cells

The ST2 status in cancer cells was evaluated for biopsy samples obtainedfrom patients by immunohistochemistry, using specific anti-human st2receptor antibody. Following the staining step the biopsy was evaluatedby a pathologist.

ImmunoCytoChemistry (ICC) of OV90 Cells Treated with dTCApFs

Materials for Cell Culture Growth and Treatment

Growing Media:

-   -   DMEM high glucose, L-Glutamine (Gibco 41965-039);    -   Sodium Pyruvate 11.0 mg/ml (100 mM) (Biological industries cat        No. 03-042-1B);    -   50 ml FBS (Biological industries cat no. 04-121-1A);    -   0.5 ml Amphotericin B 2500 μg/ml (Biological industries cat no.        03-029-1);    -   5 ml Gentamycin sulfate 50 mg/ml (Biological industries cat no.        03-035-1);        Treatment Media:        Treatment media was based on the growing media supplemented with        5% Mannitol (Sigma cat no. M4125-500 G). Media was filtered in        0.2 μm filter after adding Mannitol.        Additional Materials:    -   Trypsin EDTA (Biological industries cat no. 03-052-1B);    -   75 cm² culture Flasks (Nunc cat no. 178905);    -   25 cm² culture Flasks (Nunc cat no. 136196);    -   20 mg/ml dTCApFs in Mannitol (use aliquots, avoid repeated        freeze-thaw cycle);    -   human ovarian cancer cell lines OV90 were used: Regular OV90        (American type culture collection, ATCC) and T1/ST2 KO OV90        (manufactured by the inventors);        Materials for ICC:    -   Slides: Nunc™ Lab-Tek™ II Chamber Slide System (154534 Nunc);    -   Washing Buffer: PBS (02-023-5A Biological Industries);    -   Fixing Solution: 3% Formaldehyde (252549 Sigma) in PBS;    -   Permeabilization Solution: 0.25% Triton X-100 (0694 Amresco) in        PBS;    -   Blocking buffer: 1% BSA (A7906 Sigma), 22.52 mg/mL        glycine(G8898-500 G Sigma) in PBS with 0.1% Tween-20 (0777        Amresco);    -   Antibody buffer: 1% BSA (A7906 Sigma) in PBS with 0.1% Tween-20        (0777 Amresco);    -   Primary antibodies: anti-58k Golgi marker (ab27043) diluted        1:500, anti-β-COP (ab6323) diluted 1:250, anti-GRP78 BiP        (ab21685) diluted 1:1,000 (antibodies were purchased from        Abcam);    -   Secondary antibody: For 58k Golgi marker and β-COP: ZytoChem        Plus (HRP) Polymer anti-Rabbit (ZUC032-006). For BIP: ZytoChem        Plus (HRP) Polymer anti-Mouse (ZUC050-006);    -   DAB (3,3′-Diaminobenzidine tetrahydrochloride, D3939 Sigma);    -   Aqueous Mounting Medium (ab128982 Abcam).        Cell Culture Growth

OV90 and OV90 ST2 KO cells were grown in flasks in an incubator at 37°C., 5% CO₂ and 100% humidity until up to 70-80% density was reached. Themedium was emptied from the flasks using a pipette. Next, 4 ml TrypsinEDTA was added to each flask and the flasks were placed in the incubatorfor few minutes until most of the cells were detached from the flasks.Tapping on the flasks to increase detaching of cells was avoided. Medium(10 ml) was then added to the flasks and cultures in medium and Trypsinwere divided into 2 flasks, to each of which 15 ml fresh medium wasadded. The cells were incubated 2-3 days for growth until reaching70-80% and the passage procedure was repeated as necessary.

Cell Treatment

Cell cultures grown as detailed above in medium and Trypsin weretransferred into 50 ml conical tubes and the tubes were centrifuged at300 g for 10 min in 4° C. Then the supernatant was discarded and 15 mlfresh medium was added to the cells pellet, in order to fluidize thepellet. The cells were counted and seeded at 10,000 cells/well on achamber slide in 1 ml growing medium. The chamber slide were placed torest in the hood for 1 hr then transferred to the incubator, overnight.The following day cell treatment medium was prepared comprising 50 μg/mldTCApFs. The cells were treated by aspirating and discarding the mediumfrom the chamber slides and by adding 0.5 ml cell treatment mediumcomprising dTCApFs (or control) to each well and then incubated for 48hrs in the incubator. After that period an additional dose of treatmentmedium comprising dTCApFs was added to the treated cells wells and thecells were collected after further 24 hrs of incubation.

ImmunoCytoChemistry

Cells treated as detailed above were subjected to the following ICCprotocol. After completing of the incubation period the medium wasaspirated and the cells were washed by filling each well with washingbuffer. Next, buffer was discarded and 300 μl Fixing solution were addedto each well. Cells were incubated at RT for 15 minutes. The fixingsolution was then discarded and wells were briefly rinsed twice. Next,300 μl Permeabilization solution were added to each well and cells wereincubated at RT for 10 minutes. The Permeabilization solution wasdiscarded and wells were washed 3 times, 5 minutes for each wash.Blocking buffer (1 ml) was added to each well, and cells were incubatedat RT for 1 hr. Before the end of the incubation time, the primaryantibodies detailed above were diluted in antibody dilution buffer.Next, the blocking buffer was discarded from the wells and 120 μlprimary antibody was added to each well. The cells were then coveredwith Parafilm (or duct tape) and incubated overnight at 4° C.

The next day, the primary antibody was discarded and the wells werewashed three times with washing buffer, 5 minutes for each wash. Thesecondary antibody was then added to each well (1-2 drops) and the cellwells were incubated at RT for 30 min. Then the wells were washed 3times with wash buffer, 5 minutes for each wash. DAB substrate (freshlyprepared and filtered in 0.2 μm filter) was added to each well (1-2drops) and the cell wells were incubated 5-15 minutes while checkingdevelopment. The cell wells were washed with PBS for 5 minutes. Thewells were then removed from the slide, and the slide was air dried.Mounting media was added and the slide was put on coverslip.Visualization was performed using a light microscope.

BrdU Incorporation Assay in the Presence of Taxol or dTCApFs

Human pancreatic cancer cells (BxPC3) and human ovarian cancer cells(OV90) were treated with dTCApFs or Taxol for 24 hours in the presenceof BrdU, as detailed below.

Cells were placed at 2,000 cells/well in the middle of a 96 well platewith 100 μl DMEM (life science 41965-039) and 10% fetal calf serum (FBS,Biological industries 14-127-1A) for 24 hours in an incubator at 37° C.with 5% CO₂. The medium was then replaced to 200 μl DMEM with 2.5% FBSand Taxol (sigma T7402) at a final concentration of 2 nM or 4 nM in DMSOor dTCApFs (nextar ISK353-01 batch 351-01/1.68) at a final concentrationof 25 μg/ml in DMSO. The medium in the control cells was replaced byDMEM and FBS as indicated above. The maximal concentration of DMSO inthe cell wells was less than 0.5%. Four wells were used for eachconcentration.

Taxol- and dTCApFs-treated cells as well as the control cells werefurther incubated for 24 hours in the presence of BrdU as follows: 20 μlBrdU reagent (diluted 1:100 in DMEM supplemented with 2.5% FBS) wasadded to the cells and the plate was incubated 24 hours in 37° degreesincubator with 5% CO₂. BrdU ELISA was performed according to the kitprotocol (Millipore #2752).

BrdU Incorporation Assay in the Presence of Taxol and dTCApFs

Human pancreatic cancer cells (BxPC3) and human ovarian cancer cells(OV90) were treated with dTCApFs and Taxol for 24 hr in the presence ofBrdU, as detailed below.

Cells were placed at 2000 cells/well in the middle of a 96 well platewith 100 μl DMEM and 10% FBS for 24 hours in an incubator at 37° C. with5% CO₂. The medium was then replaced to 200 μl DMEM with 2.5% FBS anddTCApFs at a final concentration of 25 μg/ml (nextar ISK353-01 batch351-01/1.68) in DMSO. The maximal concentration of DMSO in the cellswells was less than 0.5%. Four wells were used for each concentration.Taxol at a final concentration of 2 nM or 4 nM (in 5 μl DMEMsupplemented with 2.5% FBS) was then added to the cells containingdTCApFs and the treated cells were further incubated for 24 hours in thepresence of BrdU as follows: 20 μl BrdU reagent diluted 1:100 in DMEMsupplemented with 2.5% FBS were added to the cells 24 hours before theend of experiment. Finally, BrdU ELISA was performed according to kitprotocol (Millipore #2752).

Example 1

The Peptide dTCApFs Increases Endoplasmic Reticulum (ER) Stress inCancer Cells

As indicated above, a peptide termed “T101” that is encoded by a cDNAunique to the human thymus was identified and was reported, inter alia,to reduce cancer tumor size. In addition, a peptide derivative of T101,termed herein dTCApFs (or “Nerofe”), has been reported to decrease thesecretion of proteins that are known to be associated with cancermetastasis by cancer cells and to directly inhibit migration of cancercells in vitro (16).

As described above, an ongoing clinical trial is presently performedwith the peptide dTCApFs. This peptide has the all D amino acid sequenceof Trp Trp Thr Phe Phe Leu Pro Ser Thr Leu Trp Glu Arg Lys, as denotedby SEQ ID NO: 1 and was prepared as described above.

The peptide dTCApFs was administered to cancer patients as describedabove (Table 1) at the indicated dosing regimen, intravenously (iv), 3times a week.

In order to investigate the effect of the dTCApFs peptide on cancercells, biopsies were taken from a spinal cord tumor patient treated for11 months with dTCApFs under the regimen described above. Tissuebiopsies were obtained from the patient before treatment and after 11months of treatment and were stained with anti-BiP antibody fordetecting the level of BiP, a marker of endoplasmic reticulum (ER)stress.

As detailed above, the binding immunoglobulin protein (BiP), also knownas GRP78, acts as a molecular chaperone in the ER and its synthesis ismarkedly induced under conditions that lead to the accumulation ofunfolded polypeptides in the ER.

As shown in FIG. 2, a clear difference in the level of BiP was observedin a biopsy obtained from the patient before the treatment was initiated(FIG. 2A) and after 11 month of treatment (FIG. 2B), demonstrating thatthe dTCApFs peptide increased ER stress in tumors obtained from a spinalcord neoplasm cancer patient.

Example 2

An Increase in the ER Marker BiP Level Correlates with a Reduction inTumor Size

As detailed in Example 1 above, the dTCApFs peptide was shown toincrease ER stress in tumor cells obtained from patients undergoingtherapy with this peptide, based on the observed increase in the levelof BiP. Surprisingly, the increase in the ER stress marker BiP was foundto be in a high correlation with an inhibition of tumor growth in thetreated patients, as detailed below.

The level of BiP was determined in the plasma of cancer patientsparticipating in the clinical study using the dTCApFs peptide performedas detailed above on day 1 (prior to the first administration ofdTCApFs) and on day 29 of treatment with dTCApFs. The plasma level ofBiP obtained for a cancer patient on day 1 of treatment (the “baseline”level) was then subtracted from the plasma level of BiP obtained for thesame cancer patient on day 29 of treatment.

In parallel, participating cancer patients were assessed for the size oftheir tumor using computerized tomography scan (CT), as described above,on day 1 (or just before initiation of treatment) and on last day oftreatment. The difference in the tumor size (also referred to herein as“tumor change”) was then calculated as described above. Briefly, the“tumor change” is a measure of the difference in tumor size obtainedupon treatment by dTCApFs between the first day of treatment (day 1) tolast day of treatment (duration of treatment was determined based oncriteria such as mentioned above).

Overall, 39 patients were screened, of whom 17 enrolled and completedthe study. The majority of patients (64%) were males, and the median(range) age was 65 (51-94) years. Almost half of the patients (47%) hadcolorectal (CRC) cancer and approximately a quarter (29%) had pancreaticcancer. All patients except one received several lines of anti-cancertherapy (e.g., chemotherapy, radiotherapy, biologic therapy) beforeenrolling (Table 1). The patients received 1-3 cycles of escalatingdTCApFs doses (6 mg/m², 12 mg/m², 24 mg/m², 48 mg/m², and 96 mg/m²) asdetailed in FIG. 1.

As indicated above the serum levels of the GRP78/BiP protein (as an ERstress biomarker) was measured before initiation of dTCApFs treatmentand after 29 days of treatment as indicated above. A statisticallysignificant correlation was found between the administered dTCApFs dosesand the change in serum GRP78/BiP levels (P≤0.05), as demonstrated inFIG. 3, as well as between changes in tumor size and changes in serumlevels of GRP78/BiP (P ≤0.002) (FIG. 4), suggesting that dTCApFs inducedER stress.

As shown in FIG. 4, there was a (negative) linear correlation betweenthe change in the level of BiP and the change in tumor size, namely, anincrease in the level of BiP correlated with complete inhibition oftumor growth. These results demonstrate that dTCApFs acts inter alia byincreasing the ER stress in cancer cells and without wishing to be boundby theory, via disrupting the Golgi complex, thereby leading to cancercell death.

The serum levels of BiP in patients participating in the clinical trialdetermined at days 1 and 29 of treatment as described above arepresented in Table 6 below.

TABLE 6 Serum levels of BiP in patients participating in the clinicaltrial Patient Cohort Cycle Day ng/ml BiP stDev (log) 002 1 1 1 21.300.06 002 1 1 29 26.94 2.74 006 2 1 1 9.85 6.75 006 2 1 29 38.91 3.00 0072 1 1 51.03 2.77 007 2 1 29 38.68 0.64 011 2 1 1 45.78 1.01 011 2 1 2923.89 0.44 012 3 1 1 13.81 0.55 012 3 1 29 21.17 0.23 013 3 1 1 48.330.31 013 3 1 29 26.94 3.55 015 3 1 1 56.53 2.25 015 3 1 29 46.62 1.16017 4 1 1 86.18 0.24 017 4 1 29 27.94 0.15 022 5 1 1 22.90 0.15 022 5 129 48.62 1.25 023 5 1 1 20.67 0.65 023 5 1 29 39.86 0.11 035 5 1 1 38.440.46 035 5 1 29 169.21 0.47

Interestingly, as indicated in Table 3 above, treatment with dTCApFs ata dose of 6 mg/m² led to an increase in the serum levels ofangiopoietin-1, FGF-1, FGF-2, PDGF-AA, PDGF-BB, VEGF-D, TGF-β, and VEGF.However, at doses of 12-48 mg/m², a decrease in the serum levels ofthese factors was observed, and at 96 mg/m², an increase in all factorsexcept for VEGF-D was noted. Also, serum levels of all anti-cancercytokines such as GM-CSF, IL2, IL-12p70, IL-21, and TNF-α increased withdTCApFs administration in all dose levels.

In order to explore the mode of activity (MOA) of dTCApFs, patients wereexamined by their T1/ST2 status. It was found that patients whose tumorswere T1/ST2 positive (as determined by IHC) stayed in the trial longerthan those whose tumors were T1/ST2 negative, as demonstrated in FIG. 5and experienced stable disease (SD) during dTCApFs treatment.

The T1/ST2 receptor (also referred to herein as “ST2” and “ST2/T1”) is amember of the Interleukin 1 receptors (IL-1R) superfamily. As known inthe art, members of the interleukin-1 receptor (IL-1R) superfamily arecharacterized by extracellular immunoglobulin-like domains andintracellular Toll/Interleukin-1R (TIR) domain. Members of this familyplay important role in host defense, injury and stress. It has beenpreviously reported that the thymus peptide T101, from which the peptidedTCApFs was derived, may serve as a ligand of the T1/ST2 receptor(13-15).

Therefore the levels of BiP were re-analyzed (namely the changes intumor size vs administered dTCApFs dose) for each of the populations(T1/ST2-negative positive patients and T1/ST2-positive patients). Theresults are graphically presented in FIG. 6.

As shown in FIG. 6, in which the ST2 positive and ST2 negative cancerswere separated, correlating the BiP marker level to tumor change incancer cells that were ST2 positive resulted in an R value of −0.98 andin cancer cells that were ST2 negative resulted in an R value of −0.83.The p-value of the ST2 positive graph is not optimal due to the smalldata set.

Without wishing to be bound by theory, this difference indicates thatthe higher abundance of the ST2 receptor on ST2 positive cellsfacilitates entry of the dTCApFs peptide into these cells, thuspresumably requiring lower doses of the peptide.

It is noteworthy that even in cells defined as “ST2 negative” there isample ST2 receptor for incorporating the dTCApFs peptide into the cells,albeit at a lower abundance than in the case of the ST2 positive cells.

The results presented above indicate that that the ER stress marker BiPmay be used as a marker to the efficiency of the dTCApFs peptide ininhibiting tumor growth in cancer patients treated with this peptide.Since the effect is visible already at the first month of treatment,determining the level of BiP at an early stage of treatment may serve asan evaluation test or tool for assessment of treatment efficiency and toaid in determining further treatment steps for these patients, forexample in determining whether treatment using dTCApFs should becontinued.

Example 3

Safety and Tolerability of the Peptide dTCApFs

Various parameters were analyzed for patients participating in theclinical trial referred to in Example 2 above, including safety, PK andefficacy, as detailed below.

Safety and Tolerability

Mean number of treatment cycles per patients was 3.2±1.4. Nodose-limiting toxicities (DLTs) were observed in any patient up tocohort 5. The adverse events (AEs) are summarized in Table 4 above. Nonewere related to study drug. Hypertension, anemia, vomiting, diarrhea,and abdominal pain were the most reported grade 2 AEs, and hypertensionwas the most reported grade 3 AE. Vomiting was the only grade 4 AE,reported in 1 patient. Most of the AEs were self-resolved. Overall,treatment with dTCApFs was well-tolerated with no cumulative toxicity.MTD was not reached.

Pharmacokinetics

PK results for the first day of cycles 1 and 2 are summarized in Table2; t_(1/2), Cmax and AUC0 were linearly related to dose. Dose-dependentplasma concentrations of dTCApFs were observed (FIG. 7).

Efficacy

Five of the 17 patients who were treated for ≥3 months (12, 24, and 48mg/m²) experienced stable disease (SD) throughout the treatment period.Notably, one patient was suffering from lower back pain and weakness,apparently due to a spinal cord neoplasia pressing the spinal cord,received various pain-killers drugs (e.g., tramadol, oxycodone/naloxone,morphine, and pregabalin) and used a walker. After 6 months oftreatments (12, 24, and 48 mg/m²) the patient improved her walk withoutthe need of any pain-killer medication.

Progression-free survival (PFS) analysis revealed that 6 patientsexperienced a longer PFS on dTCApFs compared to their prior regimen andone had PFS that was comparable to that on his prior regimen (these PFSvalues are indicated in Table 5 above in bold letters). In addition onepatient who did not receive prior treatments was able to stay on thestudy drug for 330 days (stained positive) (Table 5). A regressionanalysis revealed a statistically significant correlation betweenchanges in tumor size and the administered dTCApFs doses (FIG. 8).

Example 4

The Effect of the dTCApFs Peptide on ST2 Knock-Out Cells

As indicated above higher abundance of the ST2 receptor on ST2 positivecells may facilitate entry of the dTCApFs peptide into these cells. Inorder to further examine the effect of the presence of ST2 receptors oncancer cells on dTCApFs entry into cells, ST2 knock-out (KO) cells wereprepared and the levels of various proteins in these cells in responseto dTCApFs administration were examined, as detailed below.

Cells used for knock-out of the ST2 receptor were mammalian ovariancells OV90 (adenocarcinoma). As detailed above, OV90 cells and KO OV90cells were administered with dTCApFs and were then subjected to anImmunocytochemistry assay.

As evident by comparing FIG. 9A (control OV90 cells that were notadministered with dTCApFs) to FIG. 9B (OV90 cells administered withdTCApFs), dTCApFs induced complete destruction of the Golgi apparatus,resulting in ER stress. FIG. 9B shows that the Golgi disappeared, anddisrupted proteins accumulated on the ER, which is turn leads to ERstress. These results are based on analysis of the β-cop protein, whichis one of the Golgi apparatus proteins.

In contrast to the above results, as demonstrated in FIG. 9C (controlOV90 ST2 KO, not treated) and FIG. 9D (OV90 ST2 KO cells administeredwith the peptide) in OV90 ST2 KO cells, no effect of dTCApFs on theGolgi apparatus was observed (the arrows point to the intact Golgiapparatus).

FIG. 10 shows assessment of BiP expression in OV90 and in OV90 ST2 KOcells as a result of dTCApFs administration. While in OV90 cells a clearstrengthening of the BiP stain is observed due to dTCApFs treatment asshown by comparison of FIG. 10A (without dTCApFs) to FIG. 10B (in thepresence of dTCApFs), in OV90 ST2 KO cells there is no differences inthe staining of BiP as a result of dTCApFs treatment, as deduced bycomparing FIG. 10C and FIG. 10D. This means that OV90 ST2 KO cells didnot respond to dTCApFs and no ER stress was thereby induced.

Without wishing to be bound by theory, the above results demonstrate howthe ST2 receptor is related to ER stress in patients. Increasedsensitivity of ST2 positive cells to dTCApFs and in turn the aboveresults also explain the higher difference in expression levels of BiPobserved in ST2 positive cells that are demonstrated in FIG. 6.

Example 5

The Effect of the dTCApFs Peptide on Normal Cells

The peptide dTCApFs was applied to healthy human peripheral immune cellsand only minor cell death occurred, since apparently cancer cells arevery sensitive to ER stress as opposed to normal healthy cells (data notshown). Therefore, without wishing to be bound by theory, dTCApFsappears to selectively affect cancer cells.

Example 6

The Level of the ER Marker CRT does not Correlate with the AdministeredDose of the Nerofe Peptide

Calreticulin (CRT) is a chaperone expressed under normal conditions inthe ER of cells and assists in folding of newly synthesized proteins.Further to the results presented above which show an increase in the BiPER stress marker as a result of treatment with dTCApFs, changes in CRTserum levels in human patients treated with dTCApFs were also examined.Dosing patients with different doses of dTCApFs (6 mg/mm²-96 mg/mm²)induced changes in CRT serum levels, however, without any correlation tothe dose in which dTCApFs was administered, as opposed to BiP levelswhich showed linear correlation to dTCApFs administration and tumorsize, as detailed above.

A bar graph showing the serum level of CRT at the end of the treatmentby dTCApFs in cancer patients participating in the clinical studydescribed above is shown in FIG. 11A and a bar graph showing the changein serum CRT levels in patients receiving dTCApFs treatment is shown inFIG. 11B.

In summary, no correlation was observed between the dose of dTCApFs andthe level of serum CRT. In vitro experiments performed in mice showedthat CRT levels in cells were not affected due to dTCApFs treatment, asopposed to BiP, the level of which increased as a result of treatmentwith dTCApFs (data not shown).

CRT is chaperone whose level is not increased in cells when treated withdTCApFs, while the level of BiP does increase in vitro and in vivofollowing treatment with dTCApFs. Although both are part of ER stressrepair mechanism, dTCApFs selectively increases in-vitro and in-vivo ofBiP and has no influence on CRT levels. The observation that no changein CRT level occurred correlates with change of serum levels of BiP and“no-change” of CRT. This mean that BiP change in level in patientscorrelates perfect with in vitro/in vivo activity of dTCApFs.

Example 7

The Peptide dTCApFs Activates NK Cells

Natural killer cells or “NK cells” are a type of cytotoxic lymphocytecritical to the innate immune system. The role of NK cells is analogousto that of cytotoxic T cells in the adaptive immune response. Amongother functions, NK cells provide a rapid response to viral-infectedcells and respond to tumor formation.

The effect of dTCApFs was also examined on human NK cells (CD56+CD16+,purchased from Lonza (2W-501)). NK cells were seeded on LGM-3 medium(supplemented with IL-2 and IL-15). The cells were treated with dTCApFsfor 24 hours and further for 72 hours, followed by FACS analysis thatfocused on CD335 and CD337 antigens (Natural cytotoxicity triggeringreceptor 1 and Natural cytotoxicity triggering receptor 3,respectively). As shown in FIG. 12, an increase in expression of bothreceptors was induced by dTCApFs.

The CD335 and CD337 receptors are important for NK cells activityagainst cancer cells and virus infected cells. Induction of NK cellsactivity was also observed during the clinical trial described above forpatient 006 (having a spinal cord neoplasm). During the clinical trial,biopsies of patients were stained with specific anti human NK cellsantibodies before their entry to the clinical trial and after treatmentwas administered. A strong stain of NK cells in patients' biopsies afterbeing administered with dTCApFs was observed (data not shown).

Example 8

Combining dTCApFs with Taxol Results in a Synergistic Effect

The beneficial therapeutic effect of the dTCApFs peptide on cancer cellsprompted a further study, in which dTCApFs was administered to cancercells in combination with an additional anti-cancer therapeutic agent,namely Taxol, under the conditions described above.

Taxol, also known as Paclitaxel, is an anti-cancer (“antineoplastic” or“cytotoxic”) chemotherapeutic drug. Paclitaxel is classified as a “plantalkaloid,” a “taxane” and an “antimicrotubule agent” used for thetreatment of breast, ovarian, lung, bladder, prostate, melanoma,esophageal, as well as other types of solid tumor cancers.

The effect of the administered agents was monitored by aBromodeoxyuridine (BrdU) incorporation assay, used for detecting activeDNA synthesis and thereby cell proliferation and viability.

Two types of cancer cells were used for evaluating the combined effectof the dTCApFs peptide and Taxol, human ovarian cancer cells (OV90, FIG.13A) and human pancreatic cancer cells (BxPC3, FIG. 13B).

When Taxol was administered alone to human ovarian cancer cells (FIG.13A) or to human pancreatic cancer cells (FIG. 13B) no effect on cellviability was observed as compared to the control non-treated cells,based on the results of the BrdU incorporation assay performed.

In addition, as shown in Figure, 13A when the dTCApFs peptide (at 25μg/ml) was administered alone to human ovarian cancer cells no effect oncell viability was observed as compared to the control non-treatedcells.

However, when the dTCApFs peptide (at 25 μg/ml) was administered incombination with Taxol (at 2 nM) to human ovarian cancer cells, adecrease of approximately 50% in BrdU incorporation was observed (FIG.13A). This effect was enhanced for a combination of dTCApFs (at 25μg/ml) with Taxol at a concentration of 4 nM, for which no BrdUincorporation was observed.

Furthermore, as shown in Figure, 13B when the dTCApFs peptide (25 μg/ml)was administered alone to human pancreatic cancer cells, only a minoreffect on cell viability was observed as compared to the controlnon-treated cells. However, when dTCApFs (at 25 μg/ml) was administeredin combination with Taxol (at 2 nM) to human ovarian cancer cells, adecrease of approximately 20% in BrdU incorporation was observed (FIG.13B). This effect was more pronounced when a combination of dTCApFs at25 μg/ml with Taxol at a concentration of 4 nM was used, reaching areduction of over 40% in BrdU incorporation.

These results suggest a synergistic effect by the dTCApFs peptide on theactivity of Taxol, which by itself did not have any effect onproliferation, thereby allowing a reduction of the dose of Taxol used inchemotherapy. Without wishing to be bound by theory this synergisticeffect may be explained by the induction of ER stress by dTCApFs, asdemonstrated above, which contributes to promoting cell death.

Example 9

The Effect of dTCApFs and Doxorubicin on Triple Negative Breast CancerTumors

Triple Negative Breast Cancer (TNBC) is defined by the lack ofexpression of estrogen receptor (ER) and progesterone receptor (PR) andthe lack of expression or amplification of human epidermal growth factorreceptor 2 (HER2). Treatment of TNBC is presently based on a number ofagents that are approved for general breast cancer patients. However, inthe absence of specific targets for treatment, TNBC is currentlyconsidered as an aggressive cancer subtype with limited treatmentoptions and very poor prognosis following treatment with standardregimens.

The peptide dTCApFs was further assayed for treatment of TNBC asdetailed below, in combination with doxorubicin (also termed Adriamycin,Caelyx, Myocet, etc.), which is a chemotherapy currently used fortreatment of various cancer types, including breast cancer.

Therefore, 32 nude mice were inoculated subcutaneously (S.C.) with 9million human TNBC cells (human MDA231, ATCC) per mouse. Cells werecultured as known in the art, for example, as described above. When thetumors exceeded a volume of 40 mm³, the mice were randomly divided intofive groups, as follows. The “Control” group (n=5) was treated with 5%mannitol; the “dTCApFs” group (n=5) was treated once a week with dTCApFsat 15 mg/kg; the “Dox” group (n=5) was treated once a week withdoxorubicin (Sigma Aldrich) at 3 mg/kg; the “dTCApFs+Dox next day” (n=8)group was treated with dTCApFs at 15 mg/kg and 24 hours later also withdoxorubicin, at 3 mg/kg; and the “dTCApFs+Dox same day” (n=9) group wastreated with dTCApFs at 15 mg/kg and with doxorubicin at 3 mg/kg, onsame day.

As shown in FIG. 14, the combination of dTCApFs and doxorubicin,resulted in substantial attenuation in tumor volume increase in mice,either when the mice were administered with the two agents on the sameday or when mice were treated with dTCApFs and 24 hours later also withdoxorubicin. These results are significant in view of the clear increasein tumor volume observed when the mice were treated with each one of theagents alone (at the same dosing), namely either with dTCApFs or withdoxorubicin.

Furthermore, as evident from the survival curve in FIG. 15, mice treatedwith the combination of dTCApFs and doxorubicin exhibited 100% survival,either when the mice were administered with the two agents on the sameday or when the mice were treated first with dTCApFs and 24 hours lateralso with doxorubicin.

Interestingly, in mice inoculated with MDA231 TNBC cells, which arepositive for KRAS and treated with a combination of dTCApFs anddoxorubicin, under the conditions specified above, KRAS expression wasdown-regulated, as evident from comparing FIG. 16B (mice inoculated withMDA231 cells and treated with a combination of dTCApFs and doxorubicin,in which almost no KRAS fluorescence is observed) to FIG. 16A (miceinoculated with MDA231 cells, control in which KRAS fluorescence isobserved).

As known in the art, the gene KRAS has a central role in many cancertypes, including pancreatic cancer and colon cancer, etc.Down-regulation of the gene product of KRAS by the combination asdetailed above is indicative of the strong anti-cancer activity of thecombination of the invention.

Example 10

The Effect of dTCApFs and Doxorubicin on Melanoma Tumors

Melanoma, also known as malignant melanoma, is a cancer type thatdevelops from melanocytes, which are pigment-containing cells. Melanomastypically occur in the skin.

In order to examine the effect of a combination of dTCApFs anddoxorubicin on melanoma tumor volume, the following experiments wereperformed in mice. C57bl/6 mice (28) were inoculated SC with 0.2 millionB16 cells (ATCC) per mouse. B16 melanoma is a murine tumor cell lineused for research as a model for human skin cancers. When tumorsexceeded a volume of 50 mm³, the mice were randomly divided into 4groups, as follows. “Control” group (n=6) was treated with 5% mannitol;“dTCApFs” treated group (n=6) was treated once a week with dTCApFs (at15 mg/kg); “Dox” treated group (n=6) was treated once a week withdoxorubicin (at 3 mg/kg); and dTCApFs+Dox same day” group (n=10) wastreated once a week with dTCApFs and doxorubicin (at 15 mg/kg and 3mg/kg, respectively), when both agents were administered on the sameday.

As shown in FIG. 17, while tumor volume clearly increased in thepresence of each one of the agents dTCApFs or doxorubicin at the dosesindicated above, the combination of dTCApFs and doxorubicin resulted insubstantial (and synergistic) attenuation in tumor volume increase inmice (without increasing the concentration of any one of the agents inthe combination).

Example 11

The Effect of dTCApFs in Combination with an Anti PDL1 Antibody onMelanoma Tumors

As known in the art, programmed death 1 (PD-1) protein, which is aT-cell co-inhibitory receptor, and one of its ligands (PD-L1), play acentral role in the ability of tumor cells to evade the host's immunesystem. It has been previously shown that blocking the interactionsbetween PD-1 and PD-L1 enhances immune function and mediates antitumoractivity in vitro and in vivo. Therefore antibodies directed to PDL1function as immune-stimulatory agents.

The effect of dTCApFs in combination with an anti-PD-L1 antibody wasexplored in a mice model for melanoma, as detailed below. C57Bl6 mice(14) were inoculated SC with 0.2 million B16 cells per mouse. Whentumors exceeded a volume of 50 mm³, mice were divided randomly into 3groups, as follows: the “Control” group (n=5) was treated with 5%mannitol; the “anti PDL1 antibody” group (n=4) was treated with ananti-PDL1 antibody (BXcell) twice a week (at 20 mg/kg); and the“anti-PDL1 antibody+dTCApFs” group (n=5) was treated with dTCApFs and ananti-PDL1 antibody, where dTCApFs was administered three times per week,at 1 mg/kg and the anti-PDL1 antibody was administered twice a week, at20 mg/kg. The terms “anti-PD-L1 antibody” and “anti-PD-L1 antibodies”are used interchangeably.

In other words, the combination of the anti-PDL1 antibodies and thepeptide dTCApFs was administered to mice twice a week on the same day,at doses of 20 and 1 mg/kg for the anti-PDL1 antibodies and dTCApFs,respectively, and the weekly regimen also included an additional dose ofthe peptide dTCApFs, at 1 mg/kg.

As evident from FIG. 18, when mice were treated with an anti-PDL1antibody per se, there was no significant change in tumor size. However,when the anti-PDL1 antibody was combined with dTCApFs, tumor sizesignificantly decreased (without increasing the concentration of theadministered anti-PDL1 antibody).

The significant effect of the above combination on tumor size is alsodemonstrated by comparing FIG. 19A, showing a mouse having a melanomatumor that was treated with the combination of dTCApFs and an anti-PDL1antibody under the conditions specified above, to FIG. 19B, showing amouse having a melanoma tumor that was treated only with the anti-PDL1antibody. As evident from FIG. 19, the tumor volume was significantlydecreased in the presence of the above combination therapy.

Example 12

The Effect of dTCApFs in Combination with an Anti PDL1 Antibody on thePresence of Immune System Cells in the Tumor Micro Environment

The effect of administering dTCApFs in combination with anti PDL1antibodies on cells of the immune system was examined as follows.

B16F10 tumor cells were injected S.C. (200,000 cells/mice) to femaleC57BL/6 mice (JOlaHsd, 4 weeks old). When tumors reached the size of0.4-0.5 cm treatment started by administering the mice intraperitoneally(i.p.) with anti-PDL1 antibodies (in vivoMAb anti-mouse PD-L1 (B7-H1)clone 10F.9G2 Lot 615416D1 BioCell USA) on Sundays and Thursdays andwith dTCApFs (diluted in 5% mannitol) on Sundays, Mondays and Thursdays.Treatment groups were as follows: Control (treated with 5% mannitol), agroup treated with 20 mg/kg anti-PDL1 antibodies and 0.1 mg/kg dTCApFs,a group treated with 20 mg/kg anti-PDL1 antibodies and 0.5 mg/kg dTCApFsand a group treated with 20 mg/kg anti-PDL1 antibodies and 1 mg/kgdTCApFs. All of the groups were treated for 19 days.

Post treatment, the tumors were collected in 4% Formalin and after 24hours moved to 70% ethanol and then slides were prepared forimmunohistochemistry (IHC), according to cell's signaling protocol.Antigen retrieval was with Citrate at pH 6. The first antibody used fornatural killer cells detection was anti-NK (MA1-70100 thermo, 1:100diluted) and the secondary antibody used was an anti-mouse IgG (H+L),F(ab′)2 Fragment (Alexa Fluor® 488 Conjugate, #4408 1:1000 diluted). Thefirst antibody used for CD8 cells detection was anti-CD8 (ab203035abcam, 1:100 diluted) and the secondary antibody used was anti-rabbitIgG (H+L), F(ab′)2 Fragment (Alexa Fluor® 488 Conjugate, #4412 1:1000diluted).

FIG. 20A is an exemplary micrograph showing the level of NK cells intumor sections of mice treated with the combination of dTCApFs withanti-PDL1 antibodies. As evident by comparing FIG. 20A to FIG. 20C(which is a micrograph showing the level of NK cells in tumor sectionsof mice treated only with dTCApFs), the level of NK cells increased as aresult of the above combination treatment.

Furthermore, FIG. 20B is an exemplary micrograph showing the level ofCD8 cells in tumor sections of mice treated with the combination ofdTCApFs with anti-PDL1 antibodies. As evident by comparing FIG. 20B toFIG. 20D (which is a micrograph showing the level of CD8 cells in tumorsections of mice treated only with dTCApFs), the level of CD8 cells alsoincreased as a result of the above combination treatment.

Taken together the above results indicate that the combination of thepeptide dTCApFs with the anti-PDL1 antibody can increase the presence ofanticancer immune cells in the tumor. These cells can strongly inducedeath of cancer cells in the tumor.

Example 13

The Effect of dTCApFs in Combination with an Anti PDL1 Antibody onPancreatic Cancer Tumors

The effect of dTCApFs in combination with an anti-PD-L1 antibody wasfurther examined in mice inoculated with cells originating from apancreatic tumor, as detailed below. C57Bl6 mice (32) were inoculated SCwith 0.2 million Panc02 cells per mouse (ATCC). When tumor size exceededa volume of 50 mm³, the mice were randomly divided into 3 groups, asfollows: the “Control” group (n=8) was treated with 5% mannitol, the“anti-PDL1 antibody” group (n=8) was treated with an anti-PDL1 antibodytwice a week (at 20 mg/kg); the “dTCApFs” group (n=8) was treated threetimes per week with dTCApFs at 1 mg/kg; and the “anti-PDL1antibody+dTCApFs” group (n=8) was treated with dTCApFs three times perweek at 1 mg/kg and with the anti-PDL1 antibody twice per week at 20mg/kg.

As indicated above, the combination of the anti-PDL1 antibodies and thepeptide dTCApFs was administered to mice twice a week on the same day,at doses of 20 and 1 mg/kg for the anti-PDL1 antibodies and dTCApFs,respectively, and the weekly regimen also included an additional dose ofthe peptide dTCApFs, at 1 mg/kg.

As shown in FIG. 21, while in the control group and in the groupstreated with either dTCApFs or the anti-PDL1 antibody tumor sizeincreased in a time-dependent manner, when dTCApFs and the anti-PDL1antibody were administered together (as a combination), there was aslight decrease in tumor size over time, reflecting a synergistic effectbetween the two agents.

Example 14

The Effect of dTCApFs in Combination with an Anti PDL1 Antibody onBreast Cancer Tumors

The effect of dTCApFs in combination with an anti-PD-L1 antibody wasfurther examined in a mouse model of a breast cancer tumor, namely EMT6cells, as detailed below. To that end, Balb/c mice (32) were inoculatedS.C. with 0.8 million EMT6 cells (ATCC) per mouse. When the tumorsexceeded a volume of 50 mm³, the mice were randomly divided into 3groups, as follows: the “control” group (n=8) was treated with 5%mannitol; the “anti-PDL1 antibody” group (n=8) was treated twice a weekat 20 mg/kg; the “dTCApFs” group (n=8) was treated three times per weekat 1 mg/kg; and the “anti-PDL1 antibody+dTCApFs” group (n=8) was treatedwith dTCApFs three times per week at 1 mg/kg and with an anti-PDL1antibody twice a week at 20 mg/kg.

As indicated above, the combination of the anti-PDL1 antibodies and thepeptide dTCApFs was administered to mice twice a week on the same day,at doses of 20 and 1 mg/kg for the anti-PDL1 antibodies and dTCApFs,respectively, and the weekly regimen also included an additional dose ofthe peptide dTCApFs, at 1 mg/kg.

As shown in FIG. 22, while in the control group the tumor size rapidlyincreased in a time-dependent manner, the increase in tumor size wasmoderate in the groups treated with either dTCApFs or the anti-PDL1antibody, when administered as a monotherapy.

However, when dTCApFs and the anti-PDL1 antibody were administeredtogether (as a combination), there was a slight decrease in tumor sizeover time, as observed above for the pancreatic cancer cells.Apparently, dTCApFs and the anti-PDL1 antibody had a synergistic effectin attenuating tumor growth.

Taken together, the above results show that the patient may benefit fromthe combination treatment described herein, since for a dose of ananti-cancer agent that appears ineffective as a monotherapy, asynergistic beneficial effect is demonstrated when combined withdTCApFs. Lowering the dose of an anti-cancer agent may postpone drugresistance mechanisms, reduce the toxicity associated with theanti-cancer drug, etc.

Without wishing to be bound by theory, dTCApFs induces ER stressrendering the cells more sensitive to low doses of the anti-canceragents, e.g. doxorubicin, taxol and the anti-PDL1 antibodies, due toinduced expression of the protein CHOP. Once cells express CHOP theybecome very sensitive to low doses of e.g. doxorubicin. More than that,dTCApFs activates the innate immune response and in combination with theinduced ER stress in cancer cells it increases cells' sensitivity toanticancer agents.

The invention claimed is:
 1. A method of treatment of cancer in apatient in need thereof, comprising administering to said patient atherapeutically effective amount of an isolated peptide comprising theamino acid sequence denoted by SEQ ID NO. 1 or a pharmaceuticallyacceptable salt of said isolated peptide in combination withdoxorubicin, wherein said isolated peptide reduces the standard of careadministered dose of doxorubicin.
 2. The method according to claim 1,wherein the administered dose of doxorubicin is lower than the standardof care dose of doxorubicin by at least about 1%-50%, about 5%-45%,about 10%-40%, about 15%-35% or about 20%-30%.
 3. The method accordingto claim 1, wherein said isolated peptide and doxorubicin areadministered concomitantly or consecutively.
 4. The method according toclaim 1, wherein said cancer is pancreatic cancer, ovarian cancer,spindle cell neoplasm of neural origin, spindle cell neoplasm,metastatic colorectal cancer, colon cancer, colorectal cancer, colonadenocarcinoma, rectal cancer, rectal adenocarcinoma, lung cancer,non-small cell lung carcinoma, spinal cord neoplasm, breast cancer, skincancer, renal cancer, multiple myeloma, thyroid cancer, prostate cancer,adenocarcinoma, head and neck cancer, gastrointestinal cancer, stomachcancer, cancer of the small intestine, hepatic carcinoma, liver canceror malignancies of the female genital tract.
 5. The method according toclaim 1, wherein said isolated peptide consists of the amino acidsequence denoted by SEQ ID NO. 1 or a pharmaceutically acceptable saltthereof.
 6. The method according to claim 1, wherein said isolatedpeptide or a pharmaceutically acceptable salt thereof is administered ata dose of about 5 mg/m² to about 100 mg/m².
 7. The method according toclaim 1, wherein said isolated peptide or a pharmaceutically acceptablesalt thereof is administered at a frequency of once, twice or thrice perweek.
 8. The method according to claim 1, wherein said method furthercomprises administering at least one additional anti-cancer agent.